Elemental analysis of tagged biologically active materials

ABSTRACT

Methods for the detection and measurement of tagged (labeled) biologically active materials in a sample are described. The tagged biologically active materials are detected using an atomic mass or optical spectrometer having a source of atoms or atomic ions. Element-labeled biologically active materials, comprising antibodies, antibody Fab′ fragments, antigens, aptamers, protein complexes, growth factors, hormones, receptors and other biologically active materials attached to a stable elemental tag, can be used in specific binding assays and measured by elemental spectroscopic detection. Also described are methods for the determination of metals in samples of interest using specific antibodies to isolate the target metals and elemental spectroscopy for detection and quantitation. Kits are provided comprising reagents to detect and measure labeled biologically active materials or labeled competition analytes.

RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.10/614,115 filed Jul. 3, 2003, which is a continuation-in-partapplication of U.S. patent application Ser. No. 09/905,907 filed Jul.17, 2001, and claiming priority to U.S. Provisional Patent ApplicationNo. 60/258,387 filed Dec. 28, 2000.

FIELD OF THE INVENTION

The present invention relates to kits comprising reagents for thedetection and measurement of element tagged biologically activematerials and element tagged competition analytes. The present inventionalso relates to methods for the detection and measurement of elementtagged biologically active materials and further relates to thedetection and measurement of elements in a sample of interest. Moreparticularly, the present invention is directed to detection andmeasurement of tagged immunoglobulins, aptamers, antigens or analytesusing an atomic mass or optical spectrometer having a source of atoms oratomic ions.

BACKGROUND OF THE INVENTION

Various methods are in use for the detection and measurement ofbiological materials. To date, these determinations are generallyfacilitated through the use of radiological, fluorescent or enzymatictags. None of these methods have successfully dealt with elementaltagging of biologically active materials, for example immunoglobulins,aptamers or antigens, and analytes followed by detection using atomicmass or optical spectrometry.

The methods used to date have included (1) elemental tagging inimmunoassays, (2) elemental tagging using radioisotopes, (3) elementaltagging to enhance fluorescence, (4) immunological detection ofelemental species without tagging, and (5) direct elemental tagging forcell uptake studies. We will review each of these areas in turn.

Elemental Tagging in Immunoassays

Wang 1984 (U.S. Pat. No. 4,454,233) disclosed the possibility ofutilizing a mass spectrometer as a means of immunoassay detection.Wang's method required a cumbersome preliminary set of steps to firstprepare a tagged ‘mobile unit’ which was then conjugated to anantibody/antigen. In the preferred embodiment, the ‘mobile unit’ wascomprised of a latex particle embedded with heavy tagging elements suchas Fe, Ni, Cu and Co. Among Wang's reasons for utilizing ‘mobile units’were: (1) easy separation of bound reactant from unbound reactant, (2)simultaneous detection of many antigen/antibody complexes owing to thesmall size of mobile units, and (3) possible utilization of ‘unstable’or ‘reactive’ tags, as tags embedded in the latex would not interferewith the reaction. Wang's method has apparently not been accepted forimmunoassay detection.

Immunoassay detection using element tagged immunoglobulins and antigenshas also been possible using colloidal gold or extremely small beads ofgold (several nanometers in diameter), for example NANOGOLD™ particles.Van Banchet and Heppelmann (1995), Wagenknecht et al. (1994), and Wenzeland Baumeister (1995) used colloidal gold to visualize protein structurein the cell and to detect receptor-ligand binding by electronmicroscopy. However, these assays suffer from lack of quantitationcapabilities.

Element tagging has also been used in electrochemical immunoassaysfollowed by polarographic detection of the generated complexes based onthe catalytic conversion of a substrate by labeled metal ion or theanodic current of metal labeling Qiu and Song (1996). Similar to thepreceding example, the assay lacked quantitation capabilities.

Thus, although elemental tagging has been used in immunoassays, thetagging methods have been cumbersome or were ineffective atquantitation.

Elemental Tagging Using Radioisotopes

Historically, the most common use of elemental tagging has been the useof radioactive elements. While radioassays remain the method of choicedue to their exceptional sensitivity to low levels of analyte, theirgeneral use is limited by the restrictions in dealing with radioactivematerials.

Elemental Tagging to Enhance Fluorescence

Recently, elemental tagging has been used to enhance luminescence offluorescent tags. U.S. Pat. No. 4,637,988 to Hinshaw et al., describesthe use of lanthanide metals complexed with fluorescent compounds andchelating agents, that can be used in specific binding assays. U.S. Pat.No. 5,958,783 to Josel et al. describes the use of metal complexes witha charged linker as luminescent groups in fluorescence-based orelectrochemiluminescence-based assays.

However, these fluorescent tagging methods suffer from the disadvantagesassociated with their relatively low sensitivity and resulting problemswith quantitative analysis in samples containing low concentrations oftarget molecules. As well, fluorescence-based methods are limited to theanalysis and quantification of only one or at most a few targetsubstances per assay.

Immunological Detection of Elemental Species

Blake et al. (1998) and Darwish and Blake (2001) disclosed a method ofdetection and quantitation of elemental species by complexing elementalspecies with antibodies that recognize elemental species, usingantibodies conjugated with fluorescent tags. However, as outlined above,fluorescence based assays suffer from low sensitivity and are limited toone or a few targets per assay.

Direct Elemental Tagging in Conjunction with Gel Electrophoresis

Binet et al. (2001) disclosed a method of determining untagged proteinsby separation using gel electrophoresis, followed by laser ablation ofthe separated spots and detection using mass spectrometry. However, thismethod has been limited to molecules that are naturally detectable byspectrometry.

Wind et al. (2001), Nagaoka and Maitani (2000) and Baldwin et al. (2001)used chromatography to separate proteins, followed by detection usingmass spectrometry. Similarly, Chen et al, 2000 incorporated isotopictags of ¹³C, ¹⁵N and ²H in proteins before chromatographic separationand detection using organic mass spectrometry. However, separation withchromatography is an added step, which can be onerous.

Thus, if one does not tag, one is limited to what is being assayed andusing chromatography for separation adds a step to the process.

Direct Elemental Tagging for Cell Uptake Studies

Martin de Llano et al. (1996) and Martin de Llano et al. (2000),disclosed a method of visualizing and measuring the uptake of lowdensity lipoprotein (LDL) tagged with colloidal gold by cells usingelectron microscopy and mass/atomic spectrometry. However, due to theuse of colloidal particles, the difficulty in purifying labeled LDL, andthe heterogeneity of cell assay systems, absolute quantitation was notnecessary nor was it achieved.

Thus, various methods have been developed for visualizing and analyzingelement tagged biologically active compounds. However, they have innatelimitations, ranging from handling radioactive waste, to low sensitivitywith fluorescence based assays, to detection only capabilities, and tocumbersome preparation or separation steps.

Various kits are currently in use for the detection and measurement ofanalytes. These kits contain radiological, fluorescent, or enzymaticreagents and are used in conjunction with detection instruments capableof measuring absorbance, luminescence, fluorescence, chemiluminescence,or radioactivity. However, none of these kits or methods havesuccessfully dealt with the detection and measurement of element taggedbiologically active materials and analytes, in particularimmunoglobulins, aptamers, and antigens, followed by detection andmeasurement using an atomic mass or optical spectrometer.

Reagents (not currently sold in a kit format) containing element taggedimmunoglobulins are commercially available (NanoProbes). In thesereagents, the immunoglobulins are directly bound with colloidal gold orextremely small beads of gold (eg. NANOGOLD™ particles, which are 1.4 nmin diameter) and are currently used for in situ hybridization, electronmicroscopy and immunohistochemistry. In this manner, Segond von Banchetand Heppelmann (1995) and Wagneknecht et al. (1994) used colloidal goldto visualize protein structure in the cell and to detect receptor-ligandbinding by electron microscopy. In another element-tagged method,Leuvering et al. (1982) have suggested using large elemental particles(with a size varying from 10-100 nm) coated either directly onimmunological components or on inert polymer linkers and usingspectrophotometric detection to analyze reactions. However, both ofthese assays suffer from lack of quantitation capabilities.

Recently, in immunoassay kits, elemental tags have been used to enhancethe luminescence of fluorescent tags of immunoglobulins. Hinshaw et al.(1987) describe the use of lanthanide metals complexed with fluorescentcompounds and chelating agents that can be used in specificimmunoassays. Josel et al. (1990) describe the use of metal complexeswith a charged linker as luminescent groups in fluorescence-base orelectrochemiluminescence-based assays. However, these fluorescentlanthanide tag kits suffer from the disadvantages associated with theirrelatively low sensitivity and resulting problems with quantitativeanalysis in samples containing low concentrations of target molecules.As well, fluorescence-based methods are limited to the analysis andquantitation of only one or at most a few target substances per assay.

Several companies have designed kits for cytokine quantitation thatcontain radiological, fluorescent, or enzymatic reagents. For example,PerkinElmer (Wallac DELFIA™ Assay kits), BD Biosciences (OptEIA ELISA™kits), Pierce Biotechnology Inc. (Searchlight Human Cytokine™ array),R&D systems (Quantikine ELISA™ kits) and various partners of Luiminex(e.g. R&D systems, Fluorokine kits) produce kits for cytokinequantitation. However, these kits are limited to either detecting onlyone cytokine or several (4-9) over a limited dynamic range with problemsof fluorescence or chemiluminescence overlapping inhibitingsensitivities.

An enormous potential exists for the development of very simplebiological assays and kits that take advantage of capabilities offeredby elemental tagging coupled with elemental detection using a mass oroptical spectrometer. Mass and optical spectrometry offer highsensitivity, accurate quantitation and a wide dynamic range. The use ofelements to label biologically active material allows construction of anenormous number of distinguishable tags.

This invention involves bridging the science of biology, and inparticular immunology, with analytical atomic mass spectrometry. Theinvention offers an easy and simple means of tagging biologicalmolecules. Further, it offers excellent detection capabilities, equaling(or surpassing) the sensitivity of radioassays. It offers the safety offlorescence based assays, and the added feature of an enormous number ofavailable tags, with the possibility of simultaneous detection ofnumerous biological complexes. In addition, completed affinity assayscan be stored indefinitely and the handling of the reacted taggedcomplexes can be crude, as the integrity of the chemical complex neednot be preserved in assaying the element.

SUMMARY OF THE INVENTION

The last two decades have seen the improvement of elemental analysis dueto the development of the inductively coupled plasma (ICP) source usingmass or optical spectrometry. This has resulted in ultra sensitivespectrometers with high matrix tolerance and means of resolving isotopicand spectral interferences. The present invention has coupled thedevelopments in this field with the continuing need to provide rapid andprecise detection and measurement in biological assays.

In its broad aspect, the present invention provides a simple method oftagging biologically active materials, and detecting and measuring thereactant complexes by an atomic mass or optical spectrometer. Variationsof the invention include detection and measurement of elemental speciesby complexing antibodies or aptamers to elemental species, and detectionand quantitation of an analyte by tagging the analyte directly. Theinvention also provides kits comprising reagents for the detection andmeasurement of tagged biologically active materials and taggedcompetition analytes.

In addition, the present invention allows one to use a large array ofelemental tags to allow the simultaneous or sequential detection andmeasurement of biologically active material. This is known as“multiplexing”.

According to one aspect of the present invention, there is provided amethod for the detection and measurement of an element in a sample,where the measured element is a tag on a biologically active materialthat binds with one of an analyte and analyte complex, comprising: i)combining the tagged biologically active material with one of theanalyte and analyte complex, ii) separating bound tagged biologicallyactive material from unbound tagged material, and iii) detecting andmeasuring the bound tag elements by one of an atomic mass and opticalspectrometer having a source of ions or atomic ions.

According to another aspect of the present invention there is provided amethod for the detection and measurement of an element in a sample,where the measured element is a tag on a biologically active materialthat binds with one of an analyte and analyte complex, comprising: i)combining the biologically active material with one of the analyte andanalyte complex, wherein the biologically active material binds atransition element, ii) introducing the transition element to thesample, and iii) detecting and measuring the element by one of an atomicmass and optical spectrometer having a source of ions or atomic ions.

According to another aspect of the present invention, there is provideda method for the detection and measurement of an element in a sample,where the measured element is a tag on a biologically active materialthat binds with an analyte in the sample, comprising: i) directlytagging the biologically active material with a transition element, ii)combining the tagged biologically active material with the analyte inthe sample where the biologically active material binds with theanalyte, iii) separating bound tagged biologically active material fromunbound tagged material, and iv) detecting and measuring the bound tagelements by one of an atomic mass and optical spectrometer having asource of atoms or atomic ions.

According to another aspect of the present invention, there is provideda method for the detection and measurement of an element in a sample,where a primary biologically active material binds with an analyte inthe sample, and the measured element is a tag on a secondarybiologically active material that binds to the primary biologicallyactive material, comprising: i) combining the primary biologicallyactive material with the analyte in a sample, where the primarybiologically active material binds with the analyte, ii) separatingbound biologically active material from unbound biologically activematerial, iii) directly tagging the secondary biologically activematerial with a transition element, iv) introducing the tagged secondarybiologically active material into the sample where the secondarybiologically active material binds with the primary biologically activematerial, v) separating bound tagged biologically active material fromunbound tagged material, and vi) detecting and measuring the bound tagelements by one of an atomic mass and optical spectrometer having asource of atoms or atomic ions.

According to another aspect of the present invention, there is provideda method for the detection and measurement of an element in a sample,where a primary biologically active material binds with an analyte inthe sample, and a secondary biologically active molecule binds with theprimary biologically active material, and the measured element is a tagon a tertiary biologically active material that binds to the secondarybiologically active material, comprising: i) combining the primarybiologically active material with the analyte in a sample, where theprimary biologically active material binds with the analyte, ii)separating bound biologically active material from unbound biologicallyactive material, iii) introducing the secondary biologically activematerial in the sample, where the secondary biologically active materialbinds with the primary biologically active material, iv) separatingbound biologically active material from unbound biologically activematerial, v) directly tagging the tertiary biologically active materialwith a transition element, vi) introducing the tagged tertiarybiologically active material into the sample where the tagged tertiarybiologically active material binds with the secondary biologicallyactive material, vii) separating bound tagged biologically activematerial from unbound tagged material, and viii) detecting and measuringthe bound tag elements by one of an atomic mass and optical spectrometerhaving a source of atoms or atomic ions.

According to another aspect of the present invention, there is provideda method for the detection and measurement of an element of an elementalspecies in a sample, where a biologically active material specific tothe elemental species binds to the elemental species, comprising: i)introducing the biologically active material into the sample, ii)separating the biologically active material bound elemental speciescomplexes from the sample, and iv) detecting and measuring an element ofthe elemental species contained in the removed complexes by one of anatomic mass and optical spectrometer having a source of atoms or atomicions.

According to another aspect of the present invention, there is provideda method for the detection and measurement of an element in a sampleincluding an elemental species, where the measured element is a tag onan antibody specific to the elemental species, comprising: i) taggingthe antibody with a transition element, ii) introducing the taggedantibody into the sample where the tagged antibody binds with theelemental species, iii) separating tagged antibody bound elementalspecies from the sample, and iv) detecting and measuring an elementcontained in the removed complexes by one of an atomic mass and opticalspectrometer having a source of atoms or atomic ions. Variations of thisaspect include measuring and detecting the element of the elementalspecies and measuring and detecting both the tag element and element ofthe elemental species.

According to another aspect of the present invention, there is provideda method for the detection and measurement of an element in a sample,where the measured element is a tag on an analyte in a sample,comprising: i) tagging the analyte with a transition element, ii)electrophorescing the sample containing the tagged analyte, and iii)detecting and measuring the tagged analyte by one of an atomic mass andoptical spectrometer having a source of atoms or atomic ions.

According to another aspect of the present invention, there is provideda method for the detection and measurement of an element in a samplewhere the measured element is a tag on a biologically active materialthat binds with an analyte in a sample, comprising: i) binding theanalyte with a biologically active material, wherein the biologicallyactive material binds a transition element, ii) electrophorescing thesample containing the bound analyte, iii) introducing the transitionelement to the electrophoresced sample, and iv) detecting and measuringthe element by one of an atomic mass and optical spectrometer having asource of atoms or atomic ions.

According to another aspect of the present invention, there is provideda method for the detection and measurement of an element in a sample,where the measured element is a tag on a biologically active materialthat binds with an analyte in a sample, comprising: i) binding theanalyte with the biologically active material, wherein the biologicallyactive material binds a transition element, ii) introducing thetransition element to the sample, wherein the element tags thebiologically active material, and iii) detecting and measuring theelement by one of an atomic mass and optical spectrometer having asource of atoms or atomic ions. A preferred embodiment of this aspect isa method where the analyte is a cell.

According to another aspect of the present invention, there is provideda method for the detection and measurement of an element in a sample,where the measured element is a tag on a biologically active materialthat binds with an analyte in a sample, comprising: i) tagging thebiologically active material with a transition element, ii)electrophorescing the sample containing the analyte, iii) introducingthe tagged biologically active material into the electrophoresced samplecontaining the analyte, and iv) detecting and measuring the element byone of an atomic mass and optical spectrometer having a source of atomsor atomic ions.

According to another aspect of the present invention, there is provideda method for the detection and measurement of an element in a sample,where a primary biologically active material binds with an analyte inthe sample, and the measured element is a tag on a secondarybiologically active material that binds to the primary biologicallyactive material, comprising: i) electrophorescing the sample containingthe analyte, ii) introducing the primary biologically active materialinto the electrophoresced sample where the primary biologically activematerial binds with the analyte, iii) tagging the secondary biologicallyactive material with a transition element, iv) introducing the taggedsecondary biologically active material into the electrophoresced sample,where the tagged secondary biologically active material binds with theprimary biologically active material, and v) detecting and measuring theelement by one of an atomic mass and optical spectrometer having asource of atoms or atomic ions.

According to a preferred aspect of the present invention, there isprovided a method of measurement and detection of any of the aspectsdescribed above, wherein the tagged biologically active material is acommercially available product.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection of any of the aspectsdescribed above, wherein the source of atoms or atomic ions is selectedfrom a group consisting of inductively coupled plasma, graphite furnace,microwave induced plasma, glow discharge, capacitively coupled plasma,electrospray, MALDI and corona.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection of any of the aspectsdescribed above, wherein the source of atoms or atomic ions is aninductively coupled plasma source.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection of any of the aspectsdescribed above, wherein the step of detecting and measuring uses anoptical spectrometer or a mass spectrometer.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection of any of the aspectsdescribed above, wherein the element is an isotope or ion.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection of any of the aspectsdescribed above, wherein the element is selected from a group consistingof the noble metals, lanthanides, rare earth elements, gold, silver,platinum, rhodium, iridium and palladium.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection of any of the aspectsdescribed above, wherein the step of tagging involves covalentlycoupling the element to one of the biologically active material andanalyte.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection of any of the aspectsdescribed above, wherein the biologically active material is selectedfrom a group consisting of an antibody, Fab or Fab∝ fragment, aptamer,antigen, hormone, growth factor, receptor, protein and nucleic acid.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection of any of the aspectsdescribed above, wherein the tag includes more than one element.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection of any of the aspectsdescribed above, wherein the tag includes more than one isotope.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection of any of the aspectsdescribed above, wherein the tag includes more than one atom of anisotope.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection of any of the aspectsdescribed above, wherein the tag includes a different number of atoms ofeach isotope.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection described in any of theaspects above comprising an additional step of introducing two or morebiologically active materials or analytes having distinguishableelemental tags into a sample of interest for simultaneous determination.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection described in any of theaspects above comprising an additional step of sample introduction,wherein the sample introduction includes laser ablation.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection described in any of theaspects above wherein the sample introduction includes laser ablation ofpolyacrylamide or agarose gels or nitrocellulose or PVDF (polyvinylidenefluoride) or hydrophilic polypropylene (GHP, GH Polypro) membranescontaining biologically active materials or analytes tagged by at leastone element.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection described in any of theaspects above wherein the sample introduction includes laser ablation ofpolyacrylamide or agarose gels or nitrocellulose or PVDF or hydrophilicpolypropylene membranes containing biologically active materials oranalytes tagged by atoms of at least one element having an unnaturalisotopic composition.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection described in any of theaspects above wherein the sample introduction includes laser ablation ofanimal tissue samples.

According to another preferred aspect of the present invention, there isprovided a method of measurement and detection described in any of theaspects above wherein the sample introduction includes laser ablation ofcell cultures or laser ablation of the biologically active material orthe analyte.

According to another aspect of the present invention, there is provideda system of detection and measurement of molecules according to any ofthe aspects described above.

According to another aspect of the present invention, there is providedthe use of one of an atomic mass and optical spectrometer for thedetection and measurement an element described in any of the aspectsabove.

According to another aspect of the present invention, there is providedthe use of one of an atomic mass and optical spectrometer for thedetection and measurement of an elemental species described in any ofthe aspects above.

According to another aspect of the invention, there is provided a kitfor the detection and measurement of a transition element in a sample,where the measured transition element is a tag on a biologically activematerial that binds with at least one of an analyte and analyte complex,comprising a tag comprising a transition element for directly tagging abiologically active material; and instructions for i) directly tagging abiologically active material; ii) combining the tagged biologicallyactive material with at least one of an analyte and analyte complexunder conditions in which the tagged biologically active material bindswith at least one of the analyte and analyte complex, iii) separatingbound tagged biologically active material from unbound material, and iv)detecting and measuring the bound tag elements using an atomic mass oroptical spectrometer having a source of atoms or atomic ions.

According to another aspect of the invention, there is provided a kitfor the detection and measurement of a transition element in a sample,where the measured transition element is a tag on a competition analyte,comprising a tag comprising a transition element for directly tagging acompetition analyte and instructions for i) directly tagging thecompetition analyte, (ii) combining the tagged competition analyte withat least one of the analyte and analyte complex, where the taggedcompetition analyte and at least one of the analyte and anlayte complexare in competition for a binding site, iii) separating bound taggedcompetition analyte from the unbound tagged competition analyte, and iv)detecting and measuring the tag element on the bound competition analyteby an atomic mass or optical spectrometer having a source of atoms oratomic ions, wherein the detection and measurement of the tag element onthe bound competition analyte is related to the detection andmeasurement of at least one of the analyte and analyte complex.

According to another aspect of the invention, there is provided a kitfor the detection and measurement of a transition element in a sample,where the measured transition element is a tag on a biologically activematerial that binds with at least one of an analyte and analyte complexcomprising, a biologically active material directly tagged with atransition element, and instructions for i) combining the taggedbiologically active material with at least one of an analyte and analytecomplex under conditions in which the tagged biologically activematerial binds with at least one of the analyte and analyte complex, ii)separating bound tagged biologically active material from unboundmaterial, and iii) detecting and measuring the bound tag elements usingan atomic mass or optical spectrometer having a source of atoms oratomic ions.

According to another aspect of the invention, there is provided a kitfor the detection and measurement of a transition element in a sample,where the measured transition element is a tag on a competition analyte,comprising, a competition analyte tagged with a transition element, andinstructions for i) combining the tagged competition analyte with atleast one of the analyte and analyte complex, where the taggedcompetition analyte and at least one of the analyte and analyte complexare in competition for a binding site, ii) separating bound taggedcompetition analyte from the unbound tagged competition analyte, andiii) detecting and measuring the tag element on the bound competitionanalyte by an atomic mass or optical spectrometer having a source ofatoms or atomic ions, wherein the detection and measurement of the tagelement on the bound competition analyte is related to the detection andmeasurement of at least one of the analyte and analyte complex.

The kits may also comprise capture molecules that bind the analyte,analyte complex or competition analyte. The kits may also include solidsupport means, wherein the solid support means comprises binding sitesfor the analyte or capture molecules, and may include microwell platesand beads. Further, the capture molecules may include antibodies andaptamers.

The kits may also include standards, dilution buffers, elution buffers,wash buffers and assay buffers.

The element used in the kits is selected from a group consisting of thenoble metals, lanthanides, rare earth elements, gold, silver, platinum,rhodium, iridium and palladium. Further, the element may include morethan one element, isotope or atom of an isotope and may include adifferent number of atoms of each isotope.

The kits may also comprise two or more biologically active materials orcompetition analytes having distinguishable elemental tags forsimultaneous determination of two or more analytes. The kits may includereagents and instructions for two or more aspects of the presentinvention.

Another aspect of the invention is to provide a kit for the detectionand measurement of an element in a sample, where the measured element isa tag on an analyte in a sample, comprising, reagents for tagging theanalyte with a transition element, reagents for running a samplecontaining the tagged analyte on an electrophoresces gel, andinstructions for i) tagging the analyte with a transition element, ii)running the sample containing the tagged analyte on an electrophorescesgel, and iii) detecting and measuring the element by an atomic mass oroptical spectrometer having a source of atoms or atomic ions.

Another aspect of the invention is to provide a kit for the detectionand measurement of an element in a sample, where the measured element isa tag on a biologically active material that binds with at least one ofan analyte and analyte complex, comprising, a biologically activematerial that binds with at least one of the analyte and analytecomplex, a transition element, and instructions for i) combining thebiologically active material with at least one of the analyte andanalyte complex, wherein the biologically active material binds atransition element, ii) introducing the transition element to thesample, and iii) detecting and measuring the element using an atomicmass or optical spectrometer having a source of atoms or atomic ions.

The invention also provides a kit for the detection and measurement ofan element of an elemental species in a sample where an antibodyspecific to an elemental species binds to the elemental species,comprising, a biologically active material specific to the elementalspecies; and instructions for i) introducing the biologically activematerial into the sample, ii) separating the biologically activematerial bound elemental species complexes from the sample, and iii)detecting and measuring an element of the elemental species contained inthe removed complexes using an atomic mass or optical spectrometerhaving a source of atoms or atomic ions.

Another aspect of the invention is to provide a method for the detectionand measurement of a transition element in a sample, where the measuredtransition element is a tag on an aptamer that binds with an analyte,comprising combining a tagged aptamer with the analyte, where the taggedaptamer binds with the analyte, separating bound tagged aptamer fromunbound tagged aptamer, and detecting and measuring the transitionelement by an atomic mass or optical spectrometer having a source ofions or atomic ions.

Another aspect of the invention is to provide a method for the detectionand measurement of an element in a sample, where the measured element isa tag on an aptamer that binds with an analyte, comprising, combiningthe aptamer with the analyte, introducing a transition element to thecombined aptamer and analyte, wherein the transition element binds withthe aptamer, and detecting and measuring the transition element by anatomic or optical spectrometer having a source of ions or atomic ions.

Another aspect of the present invention is to provide a method for thedetection and measurement of an element in a sample, where the measuredelement is a tag on a competition analyte, comprising combining a taggedcompetition analyte with at least one of an analyte and analyte complex,where the tagged competition analyte and at least one of the analyte andanlayte complex are in competition for a binding site, separating boundtagged competition analyte from the unbound tagged competition analyte,and detecting and measuring the transition element on the boundcompetition analyte by an atomic mass or optical spectrometer having asource of atoms or atomic ions, wherein the detection and measurement ofthe transition element is related to the detection and measurement of atleast one of the analyte and analyte complex. The binding site may belocated on a capture molecule.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificpreferred embodiments of the invention are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described in relation to the drawings in which

FIG. 1 is a graph illustrating a calibration curve obtained by dilutionsof human IgG and immunoreaction with the gold-labeled anti-humanantibody with detection and quantitation using ICP-MS.

FIG. 2 is graph showing the results of an immunoassay with human IgG andFab′-Au, over a low concentration range with detection and quantitationusing ICP-MS.

FIG. 3 is a graph showing the results of an immunoassay with human IgGand Fab′-Au, over a high concentration range with detection andquantitation using ICP-MS.

FIG. 4 is a bar graph showing the results of measuring endogenousprotein in cultured cells with detection and quantitation using ICP-MS.

FIG. 5 is a graph showing the detection and quantitation of a specificanalyte by ICP-MS.

FIG. 6 is a graph showing the simultaneous detection and quantitation oftwo specific analytes (Human IgG and FLAG-Bacterial AlkalinePhosphatase, FLAG-BAP) using two different element tagged antibodies(anti-Human IgG-nanoAu and anti-mouse-Eu) in a single sample by ICP-MS.

FIG. 7 shows the results of a direct immunoassay, using ICP-MSdetection, of four human cytokines (Interferon-gamma, IFN-□; Interleukin5, IL-5; Interleukin 6, IL-6; and Interleukin 8, IL-8); simultaneouslyin pooled cytokine samples and singly in samples containing singlecytokines.

FIG. 8 shows two panels (in different scales on x-axis) of the sameexperiment in which an element-tagged sandwich assay was used to measureand quantitate two standards (provided by R&D systems and NIBSC) ofcytokine Human Interleukin 6 (IL-6) using ICP-MS detection.

FIG. 9 shows the results from two experiments in which human cytokines(Interleukin 6, IL-6 and Interleukin 8, IL-8) were measured andquantitated in buffer (1% BSA, 1× PBS) and in complex biologicalmixtures (EDTA plasma from 3 patients: I, II and III) usingelement-tagged sandwich assays with ICP-MS detection.

FIG. 10 shows the results from four experiments in which human cytokines(Interleukin 6, IL-6; Interleukin 8, IL-8; Interferon gamma, IFN□, TumorNecrosis Factor, TNF-□) were measured and quantitated in buffer (1%BSA,1× PBS) and in a complex biological mixture (EDTA plasma) usingelement-tagged sandwich assays with ICP-MS detection. Cytokineconcentrations were measured singly (red and black circles) orsimultaneously in pooled samples (triangles).

FIG. 11 shows the results of two experiments in which an element-taggedsandwich assay (using microspheres instead of a 96 well plate as asupport for the assay) was used to simultaneously measure and quantitate2 human cytokines (Tumor Necrosis Factor alpha, TNF-□ and Interleukin 6,IL-6) using ICP-MS detection. In one experiment the filtrate ofacidified microspheres was analyzed and in the second experimentunfiltered microspheres were analyzed directly by ICP-MS.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used in this application:

-   -   “analyte” means any substance being identified or measured in an        analysis, and includes but is not limited to elemental species,        element species chelate complexes; cells, viruses, subcellular        particles; proteins including more specifically antibodies,        immunoglobulins, antigens, ligands, lipoproteins, glycoproteins,        peptides, polypeptides; nucleic acids including DNA and RNA; and        including peptidic nucleic acids; oligosaccharides,        polysaccharides, lipopolysaccharides; cellular metabolites,        haptens, hormones, pharmacologically active substances,        alkaloids, steroids, vitamins, amino acids and sugars.    -   “analyte complex” means an analyte bound to other molecules or        biologically active materials.    -   “animal” means all members of the animal kingdom.    -   “aptamer” means any polynucleotide molecule (for example, DNA or        RNA molecule containing natural or synthetic nucleotides) that        has the ability to bind other molecules. For example, aptamers        have been selected which bind nucleic acids, proteins, small        organic components and even entire organisms.    -   “atomic mass spectrometer” means a mass spectrometer that        generates atomic ions, and detects atomic ions based on the        mass/charge ratio.    -   “biologically active material(s)” means any biological substance        found in nature or synthetic, and includes but is not limited to        cells, viruses, subcellular particles; proteins including more        specifically antibodies, immunoglobulins, antigens,        lipoproteins, glycoproteins, peptides, polypeptides, protein        complexes (including complexes involving ligands, receptors, or        small molecules); nucleic acids including DNA and RNA, aptamers,        and including peptidic nucleic acids; oligosaccharides,        polysaccharides, lipopolysaccharides, cellular metabolites,        haptens, hormones, pharmacologically active substances,        alkaloids, steroids, vitamins, amino acids and sugars.    -   “capacitively coupled plasma” (CCP) means a source of ionization        in which a plasma is established by capacitive coupling of        radiofrequency energy at atmospheric pressure or at a reduced        pressure (typically between 1 and 500 Torr) in a graphite or        quartz tube.    -   “capture molecule” means any molecule capable of binding with an        analyte.    -   “competition analyte” means a purified form of the tagged        analyte of interest. The tag may be a radioisotope,        fluorescence, enzyme or an element. A competition analyte is        used in competition assays in which the analyte of interest in a        sample is quantitated by determining the concentration of tagged        competition analyte that successfully binds to the analyte        binding sites provided in the assay after exposure to the sample        containing the analyte of interest.    -   “corona” means a source of ionization in which a conductor        (typically a needle) is provided a voltage relative to a counter        electrode surface (typically containing an ion sampling        aperture) such that the voltage gradient exceeds a critical        value to cause ionization of the surrounding gas, but not        sufficient to cause sparking.    -   “cytokine” means a regulator. In nature, cytokines are usually        comprised of soluble proteins and peptides that may be secreted        by one cell for the purpose of altering its own function        (autocrine effect), those of adjacent cells (juxtacrine effect),        those of near-by cells (paracrine effect), or events occurring        in the extracellular environment.    -   “elemental species” means a molecule containing a metal bound to        another atom or group of atoms. For example, selenite (SO₃ ⁻²),        selenate (SO₄ ⁻²), methylselenocysteine and selenomethionine are        elemental species of selenium.    -   “directly tagged” includes any of the methods of tagging        described herein, including but not limited to covalently and        coordinatively bound transition elements, but excluding tags        made up of encapsulated transition elements embedded in latex.    -   “element tagged” means a molecule tagged with a transition        element, including a noble metal or lanthanide.    -   “elemental tag” means any transition element, including a noble        metal or lanthanide, used to tag the biologically active        material or analyte.    -   “electrospray” means a source of ionization in which a liquid        sample is nebulized from a tube due to the sufficiently high        potential applied, which also provides a charge to the droplet,        and in which the resultant charged droplet evaporates and        fragments yielding small charged droplets or charged molecular        ions.    -   “Fab” means the antigen binding fragment of an antibody obtained        by papain reaction with immunoglobulin.    -   “Fab”′ means the antigen binding fragment of an antibody. Fab′        fragments are usually obtained by pepsin reaction with        immunoglobulin, followed by cleavage of two disulfide bonds.    -   “Glow discharge” (GD) means a source of ionization in which a        discharge is established in a low pressure gas (typically        between 0.01 and 10 Torr), typically argon, nitrogen or air, by        a direct current (or less commonly radiofrequency) potential        between electrodes.    -   “graphite furnace” means a spectrometer system that includes a        vaporization and atomization source comprised of a heated        graphite tube. Spectroscopic detection of elements within the        furnace may be performed by optical absorption or emission, or        the sample may be transported from the furnace to a plasma        source (e.g. inductively coupled plasma) for excitation and        determination by optical or mass spectrometry.    -   “inductively coupled plasma”(ICP) means a source of atomization        and ionization in which a plasma is established in an inert gas        (usually argon) by the inductive coupling of radiofrequency        energy. The frequency of excitation force is in the MHz range.    -   “lanthanide” means any element having atomic numbers 58-71. They        are also called rare earth elements.    -   “MALDI” means a source of ionization (Matrix Assisted Laser        Desorption Ionization) in which ions are produced from a sample        mixed with a matrix (typically analyzed in crystalline form) by        exposure to laser irradiation, typically at low pressure    -   “mass spectrometer” means an instrument for producing ions in a        gas and analyzing them according to their mass/charge ratio.    -   “microwave induced plasma” (MIP) means a source of atomization        and ionization in which a plasma is established in an inert gas        (typically nitrogen, argon or helium) by the coupling of        microwave energy. The frequency of excitation force is in the        GHz range.    -   “multiplexing” means using more than one elemental tag for the        simultaneous or sequential detection and measurement of        biologically active material.    -   “noble metal” means any of several metallic elements, the        electrochemical potential of which is much more positive than        the potential of the standard hydrogen electrode, therefore, an        element that resists oxidation. Examples include palladium,        silver, iridium, platinum and gold.    -   “optical spectrometer” means an instrument calibrated to measure        either wavelength of light or the refractive index of a prism,        and includes atomic emission and atomic absorption        spectrometers.    -   “plasma source” means a source of atoms or atomic ions        comprising a hot gas (usually argon) in which there are        (approximately) equal numbers of electrons and ions, and in        which the Debye length is small relative to the dimensions of        the source.    -   “primary biologically active material” means any molecule that        binds to the analyte.    -   “rare earth metals” means any element having atomic numbers        58-71. The are also called “lanthanides'.    -   “sample” means any composition of liquid, solid or gas        containing or suspected of containing an analyte.    -   “secondary biologically active material” means any molecule that        binds to the analyte or primary biologically active material.    -   “tertiary biologically active material” means any molecule that        binds to the analyte, the primary biologically active material        or the secondary biologically active material.    -   “transition element” means any element having the following        atomic numbers, 21-29, 39-47, 57-79, and 89. Transition elements        include the rare earth elements, lanthanides and noble metals.        (Cotton and Wilkinson, 1972, pages 528-530).

There are a number of aspects to the present invention.

The first aspect involves labeling of biologically active material whichbinds to an analyte in a sample. Most often, the biologically activematerial would be an immunoglobulin, aptamer or antigen. The element isdetected by an atomic mass or optical spectrometer having a source ofatoms or atomic ions. Examples 1, 2, 3, 4, 5, 6, 7, 8, 10, 14, 15 and 16are examples of this aspect of the invention.

The individual steps involved in this first aspect of the invention areknown to those skilled in the art but the coupling of assays withspectrometry is new and inventive. Each of the individual steps isdescribed in Materials and Methods section of this application.

The benefits of this aspect of the invention are that: (1) it allows forthe detection of minute quantities of analyte, (2) it allows formultiplexing, saving time, resources and providing for a better analysisof the sample, (3) the analysis is very rapid as there is no need towait for enzymatic reactions, and measurement time by ICP-OES/MS isshorter than radiological tag measurement, (4) it has a large dynamicrange, (5) radioisotopes are not required, producing a safe workenvironment and avoids toxic waste, and (6) the reacted complex does notneed to be preserved, allowing the use of acidic media to degrade thecomplex and stabilize the element in solution and thereby increasing theperiod of storage of the sample before analysis.

The second aspect involves the determination of elemental species. Incases where mass spectrometry cannot differentiate elemental species,the use of antibodies or aptamers to detect elemental species coupledwith mass spectrometry allows for their differentiation. Examples 11, 12and 13 describe this aspect of the invention in more detail. In thisaspect, as show in Example 13, multiplexing can be used. Again, thebenefits of this aspect are that: (1) it allows for the detection ofminute quantities of analyte, (2) it allows for multiplexing, (3) theanalysis is very rapid, (4) there is a large dynamic range, (5) one canavoid the use of radioisotopes, (6) the reacted complexes do not need tobe preserved, and (7) chromatographic separation is not required, whichspeeds up and simplifies the analysis.

The third aspect is the direct labeling of the analyte. The individualsteps involved in this third aspect are known to those skilled in theart, but direct labeling coupled with mass spectrometry is new andinventive. Example 17 describes this third aspect of the invention. Avariation of this aspect is described in Example 9. Again, the benefitsof this third aspect are that: (1) it allows for the detection of minutequantities of analyte, (2) it allows for multiplexing, (3) the analysisis very rapid, (4) there is a large dynamic range, (5) one can avoid theuse of radioisotopes, (6) the reacted complexes do not need to bepreserved, and (7) chromatographic separation is not required, whichspeeds up and simplifies the analysis.

The fourth aspect of the invention is the provision of kits comprisingreagents for the detection and measurement of tagged biologically activematerials and tagged competition analytes. For example, the kits mayinclude reagents for the detection and measurement of cytokines.Elemental-tagged antibodies, aptamers or cytokines in conjunction withatomic mass or optical spectrometry have never been used to quantitatelevels of cytokine in mixed biological samples.

The benefits of this aspect of the invention are many. The kits allowfor an extremely sensitive assay, there is no over-lap in signal, thereis a wide dynamic range (over 8 orders of magnitude), there is a largepotential for multiplexing (up to 167 different isotopes are available),radioisotope handling is not required, and the kits can withstand longterm storage. These benefits are discussed in detail below. The kitswill be described with reference to kits for cytokine analysis, but itis understood that kits for the analysis by mass or optical spectrometryof any analyte are within the scope of the invention.

First, the ability to quantitate multiple cytokines simultaneously withan atomic mass or optical spectrometer is not subject to the sameproblems with over-lapping signal associated with fluorescence.

Second, due to lower backgrounds and enriched tags, the sensitivity ofatomic mass or optical spectrometry in analyzing elemental tags,provides a tool that is more sensitive than existing methods. Both ofthese features allow the measurement of various cytokines in widelydifferent concentrations which is desirable when assessing immunologicalphenotypes of patients before and after different therapies.

Third, being able to determine a plurality of cytokines in a singlesample, means that less sample (e.g. blood, mucus, tissue, etc.) isrequired from each patient, which is always an advantage.

Fourth, element-tagged affinity assays can draw upon 167 differentisotopes for tags which is far more than necessary for quantitatingpools of cytokines, and this assay is therefore not limited not by thenumber tags that can be multiplexed. In comparison current kitsmanufactured that are designed for cytokine quantitation through use ofradiological, fluorescent, or enzymatic reagents are limited to eitherdetecting only one cytokine or several (4-9) over a limited dynamicrange with problems of fluorescence/chemiluminescence signalsoverlapping and inhibiting sensitivities. Fluorescent systems that claimto multiplex more than ten cytokines are not simultaneous and rely onflow cytometry to separate signals, so that the fluorometric detector isonly subjected to two fluorophores at a time. This adds to the timerequired to perform each assay as measurements are recorded bead bybead.

Fifth, the kits along with the methods provide a large dynamic range(over 8 orders of magnitude), which is not possible with eitherfluorescence or chemiluminescence. This feature is a benefit whenquantifying multiple cytokines, where it is foreseeable that onecytokine may be expressed at low levels where another may be expressedin much greater concentration.

Finally, element-tagged affinity assays permit long-term storage priorto analysis. This is very advantageous, as instrument sharing ormechanical problems can create a backlog, which means that platesdependent on fluorescence, or chemiluminescence may suffer from fadedsignals and therefore inaccurate readings. Long-term storage allows formore flexibility in both time and location as reacted plates can even beshipped dry for elemental analysis at a different location.

For all aspects of the invention, it is understood that the biologicallyactive material can be added to the analyte, or the analyte can be addedto the biologically active material. Further, an analyte complex can beformed, by the binding of molecules to the analyte, as seen in theexamples outlined below, in which a series of antibodies (primary,secondary, tertiary) can be conjugated to the analyte.

Tagging Elements

The choice of the element to be employed in the methods of the presentinvention is preferably selected on the basis of its natural abundancein the sample matrix under investigation. In order to achieveselectivity, specificity, the ability to provide reproducible results,and include appropriate standards for accurate quantitation, it isevident that the tagging element should be of low natural abundance. Forexample, in a preferred embodiment, the rare earth elements or gold canbe used as tag materials. Yet, in another embodiment, an unusual isotopecomposition of the tag can be used in order to distinguish betweennaturally present elements in the sample and the tag material. In thiscase non-radioactive isotopes of, for example, iron, potassium, nickelor sodium can be successfully distinguished from naturally abundantisotopes employing the elemental analysis.

The size of an elemental tag (ratio of atoms which are detectable bymeans of the elemental analysis to the analyte complex) may be varied inorder to produce the most consistent, sensitive and quantitative resultsfor each analyte complex.

In a preferred embodiment of this invention, several conjugates can beused in one sample simultaneously providing that the tagging materialwas selected to be different in every assay. In this embodiment thepreferred ICP-MS technique is used in order to quantify differenttagging elements simultaneously or sequentially depending on theapparatus employed.

Although many applications of the present method will involve the use ofa single elemental tag for each biologically active material (forexample, antibody, aptamer or antigen) or analyte, it should be readilyappreciated by those skilled in the art that a biologically activematerial (for example, an antibody, aptamer or antigen) or analyte maybe tagged with more than one element. As there are more than 80naturally occurring elements having more than 250 stable isotopes, thereare numerous elements, isotopes and combinations thereof to choose from.For example, there are 20 distinguishable 3-atom tags that may beconstructed from only 4 different isotopes, and one milliondistinguishable 15-atom tags that may be constructed from 10 differentisotopes, or 70-atom tags that may be constructed from 5 differentisotopes. Within limits prescribed by the need to have distinguishabletags when in combination, this will allow for simultaneous detection ofnumerous biologically tagged complexes. It is advantageous if therelative abundance of the tag elements is sufficiently different fromthe relative abundance of elements in a given sample under analysis. By“sufficiently different” it is meant that under the methods of thepresent invention it is possible to detect the target biologicallyactive material (for example, antibody, aptamer or antigen) or analyteover the background elements contained in a sample under analysis.Indeed, it is the difference in inter-elemental ratios of the taggedbiologically active material (for example, antibody, antigen or aptamer)or analyte and the sample matrix that can be used advantageously toanalyze the sample.

It is feasible to select elemental tags, which do not produceinterfering signals during analysis (i.e. do not have over-lappingsignals due to having the same mass). Therefore, two or more analyticaldeterminations can be performed simultaneously in one sample. Moreover,because the elemental tag can be made containing many atoms, measuredsignal can be greatly amplified.

Detection of Metal Ions and Elemental Species

As was indicated above, an important application of the method of thepresent invention is the detection of metal in samples, such as toxicmetals in environmental settings, including organisms, animals, andhumans. Preferably, the invention detects metals in environmentalsettings. However, as is readily apparent to those skilled in the art,the toxicity of metals depends on the oxidation state, and often on thechemical structure of the elemental species. While an elementaldetector, such as uses an ICP source, is able to determine the totalquantity of an element in a sample it is generally unable to distinguishdifferent species. There is an ongoing attempt to use different forms ofchromatography to pre-separate the sample before the ICP, but thisapproach has been plagued with concern about the integrity of thesample, i.e., preservation of the oxidation state during samplepreparation. The method of the present invention provides a means bywhich a long-standing problem of detecting speciation is overcome.

In a further embodiment of the present invention, there is provided amethod of determining the concentration of a metal ion of interest,preferably toxic metals, more preferably in environmental/biologicalsamples, comprising preparing a biologically active material (forexample an antibody or aptamer) which is specific to a selectedspeciation state of a given toxic metal, reacting said antibody with asolution suspected of containing a toxic metal, and detection of theresulting complexes by application of ICP-MS. Methods for thepreparation of an antibody which is specific to a selected oxidationstate of a given toxic metal are known by those skilled in the art andare described, for examples, in Bosslet et al.(1999), Blake et al.(1998), and Bordes et al. (1999).

In a further embodiment of the present invention, an element-taggedbiologically active material (for example, an element-tagged antibody oraptamer) is added to a sample containing a speciated element. The sampleis split into two halves. The first half of the sample is analyzed fortotal speciated element. In the second half of the sample, the reactedcomplexes are separated from the unreacted. The tagging element and thespeciated element are quantified in the reacted sample. The speciatedelement is also quantified in the unreacted sample. In this instance,the results will provide complementary data, and the fraction of thespecific species in question will be determined.

As was also indicated above, an important application of the method ofthe present invention is the detection of elements of tags in samples bymeans of laser ablation of polyacrylamide gels where tagged moleculesare separated by electrophoresis. Optionally, the sample can be run onan electrophoresis gel and then probed using element tagged biologicallyactive materials, for example antibodies or aptamers. This applicationcan be used in order to analyze biomolecules in gels or membranesrapidly without destroying the sample. Also, by employing microablationit is feasible to distinguish cancerous cells from normal cells onhistological section of biopsy samples using element-tagged antibodiesspecifically attached to the markers of cancerous populations.

The following section describes the methods and materials required tocarry out the following invention.

Methods and Materials ICP-MS Techniques

Techniques using ICP-MS or OES can be applied for the purposes of thisinvention.

For example, in its latest realization it was described in Tanner elal.(2000a), Baranov et al. (1999), Tanner et al. (1999), Tanner et al.(2000b), and Bandura et al. (2000). This successful modification ofICP-MS includes the dynamic reaction cell, which is used in order toreduce isobaric interferences in atomic mass spectrometry. Briefly, theICP-DRC-MS technique comprises a high temperature plasma in which thesample particles are atomized and ionized; vacuum interface which isdesigned to transport the plasma together with analyte ions fromatmospheric pressure to vacuum; ion focusing optics; the dynamicreaction cell for chemical modification of the ion current and massanalyzing devise (quadrupole, TOF or magnetic sector). The sample isusually introduced to the plasma as a spray of droplets (liquid sample)or flow of particles (laser ablation of solid surfaces).

Sources of Atoms and Atomic Ions

The source of atoms or atomic ions can be produced from the followingsources: inductively coupled plasma (ICP), graphite furnace, microwaveinduced plasma (MIP), glow discharge (GD), capacitively coupled plasma(CCP), electrospray, MALDI or corona.

Antibody Preparation

According to a preferred embodiment of the methods of the presentinvention, elementally tagged antibodies, or antibodies directed to ametal of interest are employed. Antibodies that bind a target ofinterest can be prepared using techniques known in the art such as thosedescribed by Kohler and Milstein (1975), Wakabayashi et al. (1990),Frackelton et al. (1985) and Gillis (1983), which are incorporatedherein by reference. (See also Kennett, McKearn, and Bechtol (1980), andHarlow and Lane (1988), which are also incorporated herein byreference).

Within the context of the present invention, antibodies are understoodto include monoclonal antibodies, polyclonal antibodies, antibodyfragments (e.g., Fab, and F(ab′)₂) and recombinantly produced bindingpartners. Antibodies are understood to be reactive against the targetanalyte if they bind to the target with an affinity of greater than orequal to 10⁻⁶ M.

Aptamer Preparation

According to a preferred embodiment of the methods of the presentinvention, elementally tagged aptamers directed to an anlayte areemployed. Aptamers that bind a target of interest can be prepared usingtechniques known in the art such as those described in Ellington A D andSzostak J W. (1990); Turek C and Gold L (1990); Robertson D L and JoyceG F (1990); Gold, L, Polisky, B, Uhlenbeck, O, and Yarus, M (1995);Szostak, J W (1995).

Tagging of Biologically Active Materials

Preferably, the tagging element is in the form of a nanoparticle, whichis attached to a biologically active material, such as for example anantibody, without degrading its activity (tagged conjugate). Examples oftechniques for coupling elemental tags to biologically active materialsare well known to those skilled in the art. For example, Barlett, P. A.et al. (1978), describe a metal cluster compound (Au₁₁) having a core of11 gold atoms with a diameter of 0.8 nm. The metal core of 11 gold atomsin the undecagold metal cluster compound is surrounded by an organicshell of PAr₃ groups. This metal cluster compound has been used to formgold immunoprobes, for example, by conjugating Au₁₁ to Fab′ antibodyfragments as well as other biological compounds.

Another metal cluster compound which has been used as a probe isNanogold™. Nanogold™ has a metal core with 50-70 gold atoms (the exactnumber not yet being known but believed to be 67 gold atoms) surroundedby a similar shell of organic groups (PAr₃) as undecagold. The metalcore of Nanogold™ is 1.4 nm in diameter.

A more recent description of techniques for the preparation ofbiological tags, which may be used in the method of the presentinvention, is found in Hainfeld et al. (1996) (U.S. Pat. No. 5,521,289).Briefly Hainfeld et al. (1996) describes, among others, thiol goldclusters produced by forming an organic-gold complex by reacting acompound containing a thiol with gold in solution. A second equivalentis also added of the thiol compound. Finally the gold organic is reducedwith NaBH₄ or other reducing agents and organometallic particles areformed. These have the general formula Au_(n)R._(m)R′₁ . . . , where n,m, and 1 are integers, R and R′ are organic thiols, (e.g., alkyl thiols,aryl thiols, proteins containing thiol, peptides or nucleic acids withthiol, glutathione, cysteine, thioglucose, thiolbenzoic acid, etc.).With two equivalents of organic thiol compound, clusters with gold cores˜1.4 nm are formed with many organics. The organic moiety may then bereacted by known reactions to covalently link this particle toantibodies, lipids, carbohydrates, nucleic acids, or other molecules toform probes. Mixtures of organic thiols may be used to provide mixedfunctionality to the clusters. These organo-gold clusters are stable toheating at 100 degrees C.

These organic thiol-gold preparations may also be made using similarprocesses with alternative metals to gold, e.g., platinum, silver,palladium and other metals, or mixtures of metal ions, e.g., gold andsilver, resulting in mixed metal clusters. The metal clusters togetherwith all other components of a sample are readily atomized and ionizedin the high temperature ICP for subsequent MS or OES analysis.

Separation Techniques

According to one embodiment of the present invention, a tagged conjugatemay be isolated for analysis by employing a filtration technique. Forexample, after incubation of an analyte with the tagged conjugate thesample undergoes filtering through a size separating centrifugal filter.Non-reacted tagged antibody together with other components of the samplemixture including non-reacted antigen pass through the filter into thefiltrate. Complexes of analyte and antibody conjugate are left on thefilter and after washing can be stabilized in acidic solution. Since theintegrity of the sample (i.e. the chemical form) is not important afterseparation, the separated sample can be acidified/degraded/stabilized(for example in acidic media) and quantitative analysis is preferablycarried out using the ICP-MS technique. The optimal concentrations ofall reagents for each system should be determined in an initialcriss-cross serial dilution experiment and the concentration of reagentbeing quantitated must lie within the dynamic range of the standardcurve. As will be readily apparent to those skilled in the art, othertechniques of separation of free substance or non-complexed proteinsfrom complexed substance may be used, for examples, salting out,chromatography, electrophoresis, gel filtration, fractionation,absorption, polyacrylamide gel electrophoresis, agglutination, affinityseparations, immunoassays, or combinations thereof.

Kits

Kits are provided for all aspects of this invention.

Kits are provided for the first aspect of the invention in which abiologically active material binds to an analyte in a sample. Examples1, 2, 3, 4, 5, 6, 7, 8, 10, 14, 15 and 16 describe this aspect of theinvention. The kits may include 1) tags comprising transition elements;2) element-tagged biologically active materials (including antibodies,aptamers, antigens, or combinations of the above) or element-taggedcompetition analytes, 3) solid supports, for example microwell plate orbeads and filter-plate, 4) analyte standards (i.e. analytes of knownconcentration, unlabeled in the sandwich and direct assays andelement-labeled in the competition assays), 5) diluent buffers, 6) assaybuffers, 7) wash buffers, 8) elution buffers and 9) protocols andinstructions to carry out the detection and measurement of an element ina sample.

Each of these components is discussed below.

(1) The tags comprising transition elements as described above.

(2) The element-tagged biologically active material would preferablycontain element tags that are biologically inert and uniform in bothsize (number of atoms) and isotopic purity. Preferably the tagging ofthe biologically active material involves the covalent attachment ofelemental tags to the biologically active material at sites thatminimize loss of activity. The element-tagged biologically activematerial may include any biologically active material, for example,antibodies, aptamers, or antigens.

In kits designed for competitive assays, competition analytes ofinterest are tagged with elements. The tags are preferably biologicallyinert and uniform in both size (number of atoms) and isotopic purity.Preferably they are water soluble, non-toxic, easily separated from atagged material by known chromatographic or dialysis methods.

In the preparation of element-tagged biologically active materials orcompetition analytes, purification steps are required to separate freeelement tags from tagged biologically active materials or competitionanalytes. This may be done using size exclusion chromatography, affinitychromatography, filtration, or dialysis. The purity and quantity of theelement-tagged biologically active materials or competition analytes canbe analyzed through UV spectrophotometry or atomic mass or opticalspectrometry. The affinity of the element-tagged biologically activematerials and the element tagged-competition analytes to the targetmolecule is determined by test protocols of element-tagged immunoassayprior to sale of the kit.

(3) The assay may comprise solid supports such as microwell plates,beads columns, filters, membranes, gels, or sol-gel to support sandwich,direct, or competitive element-tagged affinity assays. The beads maycomprise agarose, sepharose, polystyrene, or polymeric micro spheres.

(4) Purified standards are provided in the kit for the analytequantitation and consist of known concentrations of purified analyte(unlabeled for sandwich or direct assay and element-labeled forcompetition assay) and enable calibration curves to be prepared. Thestandards should be free of high mass elements or chelators that wouldinterfere with element analysis and are preferably lyophilized in asterile buffered protein base with a preservative.

(5) Diluent (or dilution) buffers may also be provided for dilution ofthe purified element-tagged biologically active materials,element-tagged competition analyte, standards or the analyte. Diluentbuffers are preferably: sterile, free of high mass elements orchelators, non-toxic, and designed to retain solubility, bindingaffinities, and native forms of element-tagged biologically activematerials, element-tagged competition analyte, and the analyte to beanalyzed. The buffers may comprise sterile proteinaceous buffers withpreservatives and free from high mass elements or chelators. Diluentbuffers for aptamers should be DNase and RNase free and may requireDNase or RNase inhibitors to prevent aptamer degradation. Suggesteddilution factors may also be included.

(6) The assay buffers are used to pre-treat the support prior to theassay. These buffers will serve to pre-wet, optimize the pH, chemicallyactivate the supports to be used, or any combination of the above. Assaybuffers are preferably: sterile, free of high mass elements orchelators, non-toxic, and designed to retain solubility, bindingaffinities, and native forms of element-tagged biologically activematerials and element tagged competition analytes to be analyzed.

(7) The wash buffers are also included in the kit for washing excessunbound element-tagged reagent from the assay. These buffers arepreferably: sterile, free of high mass elements or chelators, non-toxic,and designed to retain solubility, binding affinities, and native formsof element-tagged biologically active materials, element-taggedcompetition analytes, and the analyte to be analyzed.

(8) The elution buffer is used to suspend the element tag from thesurfaces of microwell plates or beads to allow for introduction into anatomic mass or optical spectrometer and should be free of high masselements with the exception of an elemental internal standard of knownconcentration. The tag does not necessarily have to be separated fromthe biologically active material or the beads. The elution buffer maypreferably be an acid that allows complete solubility of the sample(tag, analyte, antigens, proteins, and aptamers) and separation fromplate or bead support. Preferably the elution buffer is also spiked withan element or enriched isotope that has not been used as a tag. Thespike will allow for monitoring of any instrumental drift. The elutionbuffer may not be required for microsphere assays, in which case,reacted microspheres may be suspended in wash buffer and introduceddirectly into the atomic mass or optical spectrometry.

(9) Instructions or protocols may also be included for conducting theassays according to the methods described in the invention.

Kits are provided for the second aspect of the invention for thedetermination of elemental species. Examples 11, 12 and 13 describe thisaspect of the invention. The kits may include 1) a biologically activematerial (for example, an antibody or apatmer) specific to the elementalspecies, 2) buffers as described above and 3) instructions for carryingout the protocols as described herein.

Kits are also provided for the third aspect of the invention thatinvolves direct labeling of the analyte. Examples 9 and 17 describe thisaspect of the invention. The kits may include 1) a tag comprising atransition element, 2) reagents for tagging the analyte with atransition element as is known to those skilled in the art, 3) reagentsfor running a sample containing the tagged analyte on anelectrophoresces gel, 4) buffers as described above and 5) instructionsfor carrying out the protocols as described herein.

In light of the present disclosure, those skilled in the art willreadily appreciate other methods and applications of the methods of thepresent invention.

The examples below are non-limiting and are merely representative ofvarious aspects and features of the present invention.

EXAMPLES Example 1 Nanogold™ Immunoassay

The following provides an example of the methods of the invention usingthe Nanogold-IgG™ (or Nanogold-Fab′™; Nanoprobes) immunoassay and itsprotocol. PBS buffer A is prepared as follows: 15 mM NaCl; 2 mM sodiumphosphate, pH 7.4; 1% BSA (bovine serum albumin). All eppendorf tubes,micro-titer plates, and filters to be used subsequently are treated withPBS buffer A for 1 hour at room temperature to block non-specificbinding. This treatment will reduce the non-specific interactions thatoccur between the plastic used and the analyte and Nanogold-IgG™.Alternatively low retention plastic products can be used (e.g. Axygentubes). Following this a solution of the analyte (peptide, protein,etc.) in the concentration range of 1000 to 0.5 pg/μl in PBS buffer A isprepared. All dilutions are stored on ice. Subsequently, 100 μl ofeither analyte dilution or PBS buffer A (for controls) is pipetted intothe individual wells of the micro-titer plate (or set of eppendorftubes). The Nanogold-IgG™ is pre-filtered through 300 kDa (MICRCON™ orCentricon™) centrifugal filter devices. Dilutions of filteredNanogold-IgG™ in PBS buffer A are prepared as follows: A 1:50 dilutionis produced by adding 60 μl of Nanogold-IgG™ in 2940 μl PBS buffer A. A1:500 dilution is then produced by adding 100 μl of 1:50 Nanogold-IgG™to 900 μl of PBS buffer A. Depending on concentration range of analyte,100 to 500 μl of 1:500 Nanogold-IgG™ is then added to the wells of theplate and then incubated for 1-2 hours at room temperature. The totalamount of analyte-Nanogold-IgG™ mix is then pipetted into the samplereservoir (upper chamber) of a 300 kDa MICROCON™ centrifugal filterdevice (max volume 2 ml). This sample is centrifuged at 14,000 g for 15minutes at room temperature. The assembly is removed from the centrifugeand the vial separated from sample reservoir. The sample reservoir isinverted in a new vial, and spun for 3 minutes at 1000 g to transfer theconcentrate to a new vial. Finally, a fixed volume of the collectedanalyte-Nanogold-IgG™ antibody mixture is diluted to 1 ml with 10% HCl/1ppbIr for stabilization. Ir provides an internal standard for ICP-MSquantitation and the acid solution is suitable for the elementalanalysis. The linearity of the ICP-MS detector response as a function ofthe concentration of the analyte human IgG is shown in the resultspresented in FIG. 1.

Example 2 Immunoassay, Other than Nanogold-IgG™ and Assay with Aptamers

According to this example, an antibody is tagged with an element (eg.Eu, Ru, etc.) suitable for analysis by ICP-MS and is introduced into asample containing an analyte which is an antigen of interest (e.g. humanblood proteins). The element-tagged antibody reacts specifically to thetarget analyte. The resulting tagged analyte complex is separated fromun-reacted antibody (as in Example 1, 3, 4, 5, 8, 9, or 10), and thetagged complex is analyzed by ICP-MS. Variations of this exampleinclude:

-   -   a) Tagging with multiple atoms to amplify the signal and thereby        improving detectability.    -   b) As, a), except the tag contains several isotopes of the same        element or different elements, preferably in a non-natural        (unusual) distribution, so that the unique isotope distribution        is a determinant of the targeted analyte. It is to be recognized        that there are more than 80 naturally occurring elements) of        which some 60 may have value in this application) having more        than 250 stable isotopes. This allows construction of an        enormous number of distinguishable tags. For example, there are        20 distinguishable 3-atom tags that may be constructed from only        4 different isotopes, and one million distinguishable 15-atom        tags from 10 different isotopes, or 70-atom tags from 5        different isotopes.    -   c) As in a) and b), but incorporating different antibodies with        specificity to different target molecules, to allow simultaneous        determination of different target molecules. The number of        simultaneous determinations is limited by the number of        distinguishable tags in combination (which is fewer than the        number of distinguishable tags in isolation as described above).    -   d) Using aptamers, Fab′, Fab groups in place of antibodies. This        is useful when the analyte that you are interested in binding or        quantitating is too small or too toxic to have an antibody made        that will bind efficiently to it.

Example 3 Protein A Sepharose Immunoassay

The following provides an example of the methods of the invention usingthe Protein A Sepharose CL-4B™ (Pharmacia) immunoassay and its protocol.Either Nanogold-Fab′™ or another element-labeled Fab′ specific to thetarget analyte (or host species of the secondary antibody) may be used.There are three types of immunoassays that may be used:

-   -   a) Direct immunoassay, which would involve trapping the target        protein of interest (protein X) by incubating Protein A        Sepharose CL-4B™ with an excess of antibody specific to the        target analyte, washing off the un-reacted antibody, adding the        analyte-containing sample, washing off the unbound components,        and then exposing the PAS-antibody-protein X complexes to        element-labeled, anti-X Fab′. This is also referred to as a        sandwich assay.    -   b) Indirect immunoassay which would involve trapping the target        protein of interest (protein X) by incubating Protein A        Sepharose CL-4B™ with an excess of primary antibody (e.g.        polyclonal) specific to the target analyte, washing off the        un-reacted primary antibody, adding the analyte-containing        sample, washing off the unbound components, and then exposing        the PAS-antibody-protein X complexes to a second antibody        specific to protein X (e.g. a monoclonal antibody), washing off        un-reacted secondary antibody, and then incubating the        PAS-antibody-protein X-antibody complexes with an        element-labeled, anti-secondary Fab′. Alternatively beads or        micro-titer plates covalently bound to anti-protein X antibodies        may be used. This is also referred to as an indirect sandwich        assay because proteins are anchored to a surface using a capture        molecule.    -   c) Competition immunoassay, which would involve trapping the        target protein of interest (protein X) by incubating Protein A        Sepharose CL-4B™ with an excess of antibody specific to the        target analyte, washing off the un-reacted antibody, adding the        analyte-containing sample, washing off the unbound components,        and then exposing the PAS-antibody-protein X complexes to a        known amount of purified element-labeled protein X.        PBS buffer A is prepared as follows: 150 mM NaCl; 20 mM        phosphate, pH 7.4; 1% BSA (bovine serum albumin). All eppendorf        tubes, micro-titer plates, and filters, and slurry of Protein A        Sepharose CL-4B™ to be used subsequently are treated with PBS        buffer A for 1 hour at room temperature to block non-specific        binding. This treatment will reduce the non-specific        interactions that occur between the plastic used and the analyte        and Nanogold-Fab′™. Alternatively low retention plastic products        can be used (e.g. Axygen tubes). Following this a solution of        the analyte (peptide, protein, etc.) in the concentration range        of 1000 to 0.5 pg/μl in PBS buffer A is prepared. All dilutions        are stored on ice. Subsequently, 100 ul of either analyte or PBS        buffer A (for controls) is pipetted into the individual wells of        the micro-titer plate (or set of eppendorf tubes). To remove        large unbound gold particles, the Nanogold-Fab′™ is pre-filtered        through 300 KDa MICRCON™ (or Centricon™) centifugal filter        devices. Dilutions of filtered Nanogold-Fab′™ in PBS buffer A        are prepared as follows: A 1:50 dilution is produced by adding        60 μl of Nanogold-Fab′™ in 2940 μl PBS buffer A. A 1:500        dilution is then produced by adding 100 μl of 1:50        Nanogold-Fab′™ to 900 μl of PBS buffer A. Depending on        concentration range of analyte, 100 to 500 μl of 1:500        Nanogold-Fab′™ is then added to the wells of the plate and then        incubated for 1-2 hours at room temperature. The sample is        centrifuged at 14,000 rpm for 2 minutes at room temperature. The        beads are washed four times with PBS buffer A. In method b) the        additional steps to include consist of incubating the beads (and        attached analyte) with unlabeled primary antibody, washing off        un-reacted monoclonal antibody, and then incubating the        PAS-antibody-protein X-antibody complexes with an        element-labeled, anti-X Fab′. Finally, a fixed volume of 10%        HCl/1 ppbIr is added to each well. Ir provides an internal        standard for ICP-MS quantitation and the acid solution is        suitable for the elemental analysis.

Experimental results obtained according to method a) are given in FIGS.2 and 3, using human IgG as the analyte with F′ab-Au as the taggedantibody. FIG. 2 provides the calibration results over a relatively lowconcentration range, and FIG. 3 over a higher concentration range.Together, the data exhibit greater than 3 orders of magnitude ofdetector linearity with respect to the analyte concentration.

This Example also permits multiplexing to be analyzed and can be used toidentify protein-protein interactions. In this method, cell lysate iscollected and subjected to the method as above where an interaction issuspected between protein A and protein B. In this case the primaryantibody would be specific to protein A and an element-labeled Fab′would be specific to protein B. Interactions with multiple otherproteins (e.g. protein C and protein D) could be detected at the sametime, providing that different elements were used to label anti-Fab′specific to protein C and anti-Fab′ specific to protein D.

Example 4 Dynabeads™ Immunoassay

The following method provides an example of the invention using theDynabeads™ (Dynal) immunoassay and its protocol. This immunoassay isperformed as in Example 3, using Dynabeads™ in place of Protein ASepharose CL-4B™. Instead of centrifuging the sample, the sample isexposed to a magnetic device (Dynal MPC™). This draws the beads to thebottom of the wells between and after each wash step. Again, 10% HCl/1ppbIr is added to each well in the final step to provide an internalstandard for ICP-MS quantitation and elemental analysis.

In the same manner as described for Example 3, multiplexing andprotein-protein interactions can be identified using this method.

Example 5 Method for Detection and Quantification of Endogenous Proteinsin Cultured Cells

There are two methods by which the discrete changes in the levels ofendogenous proteins in culture cells can be measured.

-   -   a) Direct immunoassay, in which an antibody specific to the        protein of interest is required.

This antibody is labeled with an element suitable for analysis byICP-MS.

-   -   b) Indirect immunoassay, in which an antibody (primary antibody)        specific to the protein of interest is required. In addition a        secondary antibody specific to the primary antibody is labeled        with an element suitable for analysis by ICP-MS.

A mono-layer of attached cultured cells is grown and treated withconditions of interest. The growth media is removed and the cells arewashed with 1× PBS three times. PBS is then replaced with ice-coldmethanol and the culture dishes are incubated at −20° C. for 5 minutes.The methanol is removed and the cells are allowed to dry completely. Anassay buffer (e.g. 10% horse serum, 1% BSA, 0.05% Tween-20, 1× PBS) isadded to the culture dishes and the dishes are incubated for 1-2 hoursat room temperature. In method a) an antibody specific to the protein ofinterest is labeled with an element, diluted in dilution buffer andadded to the culture dishes. The cells are exposed to the antibody mixfor 2 hours at room temperature (or 37° C.). The un-reacted primaryantibody is washed away with wash assay buffer. During this time, theelement-tagged antibody binds the target protein. In method b) theantibody specific to the protein of interest is not labeled and isdiluted in dilution buffer and added to the culture dishes. The cellsare exposed to the antibody mix for 2 hours at room temperature (or 37°C.). In the next step, the un-reacted primary antibody is washed awaywith wash buffer. In method b) the element-labeled secondary antibody isdiluted in assay buffer and applied to the cells. The dishes areincubated for 1-2 hours at room temperature. The un-reacted secondaryantibody is then washed away with wash buffer. Finally, in both methods,an acid solution (e.g. concentrated HCl) is added, to release anddissolve the tagging element. The dissolved element in acid is dilutedwith 10% HCl/1 ppb Ir to provide an internal standard. The acid solutioncontaining the tagging element is then analyzed by ICP-MS to quantifythe protein of interest.

Experimental data obtained according to method b) is shown in FIG. 4.This data examines the sensitivity of this immunoassay, by comparing therelative amounts of Smad2 in three different cell cultures; COS (Bars 5and 6), COS transfected with pCMV5B-Smad2 (COS-smad2) (Bars 1 and 2),and C2C12 cells (Bars 3 and 4). COS cells are known to have undetectablelevels of Smad2 protein (using Western blot analysis). Conversely, Smad2is detectable in C2C12 cell lysate and in COS cells that have beentransfected with pCMV5B-Smad2. These cell cultures are prepared in 60 mmdishes, fixed with methanol, blocked with TBST buffer and then incubatedin either the presence (Bars 2, 4, and 6) or absence (Bars 1, 3, and 5)of polyclonal anti-Smad2 antibody (Upstate Biotech). The cells are thenincubated with a gold-tagged anti-rabbit antibody (Nanoprobes),dissolved in concentrated HCl, diluted 2 fold in 10%HCl/1 ppb Ir andanalyzed using the ICP-MS. Each bar is an average of triplicate samples.Bars 1, 3, and 5 reflect negative control cultures not treated withprimary antibody (∓). Cultures treated with both primary and secondaryantibodies (++) show that in the two cell cultures that express Smad2, asubstantial increment in the signal over the (∓) results indicates thepresence of the Smad2 protein. The third culture, COS, which is notexpected to express Smad2, shows a signal for the (++) case that isroughly comparable to that of the blank (∓).

Example 6 Method for Determination Efficiency of Cell Transfection

The effectiveness of the cell culture transfection is determined byfirst modifying cells to transduce a tail (e.g. FLAG™). This protocol isuseful when antibodies against the analyte of interest are notavailable. In this case, the expression of a recombinant analytecontaining recognizable tails such as Flag, HIS (histidine) or GST(glutathione S-transferase) are particularly useful as antibodiesagainst these moieties are readily available. As in Example 5, there aretwo methods by which the analyte of interest can be detected (directlyand indirectly).

-   -   a) Direct immunoassay, in which an antibody specific to the tail        is required. This antibody is labeled with an element suitable        for analysis by ICP-MS.    -   b) Indirect immunoassay, in which an antibody (primary antibody)        specific to the tail is required. In addition a secondary        antibody specific to the primary antibody is labeled with an        element suitable for analysis by ICP-MS.

Between 1-3 days after transfection of the cells, the growth media(typically 10% FBS, depending on cell-type) is removed and themono-layer of attached cells are washed with 1× PBS three times. PBS isreplaced with ice-cold methanol and the culture dishes are incubated at−20° C. for 5 minutes. The methanol is removed and the cells are allowedto dry out completely. An assay buffer (e.g. 10% horse serum, 1% BSA,0.05% Tween-20, 1× PBS) is added to the culture dishes and the dishesare incubated for 1-2 hours at room temperature. In method a) anantibody specific to the tail is produced and labeled with an elementthat is suitable for analyzing with the ICP-MS. The antibody is dilutedin dilution buffer and added to the culture dishes. The cells areexposed to the antibody mix for 2 hours at room temperature (or 37° C.).During this time, the element-tagged antibody binds the target proteinthrough its tail. In method b) the antibody specific to the protein ofinterest is not labeled. In both cases, the un-reacted primary antibodyis washed away with wash buffer. Then, in method b) the element-labeledsecondary antibody is diluted in dilution buffer and applied to thecells. The dishes are incubated for 1-2 hours at room temperature. Theun-reacted secondary antibody is washed away with wash buffer. Finally,in both methods, an acid solution (e.g. concentrated HCl) is added, torelease and dissolve the tagging element. The dissolved element in acidis diluted with 10% HCl/1 ppb Ir to provide an internal standard. Theacid solution containing the tagging element is analyzed by ICP-MS toquantify the efficiency of the transfection. Culture dishes containingnon-transfected cells cultured at the same time can be used as anegative control An alternate variation of this Example involves using a6× HIS-tagged construct™ (Invitrogen), where there is no need foranalyte-specific antibodies. Cells transfected with 6× HIS-taggedconstructs are fixed with methanol, blocked with the assay buffer andincubated for 2 hours with a solution containing nickel (e.g. Ni—NTA™;Qiagen). The cells are washed to remove free nickel, degraded in aciddegraded, and analyzed using ICP-MS for nickel content.

Example 7 Reporter Assay

In the study of transcription factors, it is necessary to quantitate thelevels of transcription. There are two methods by which discrete changesin the levels of transcription activity on a specific promoter (orenhancer elements) can be measured. Cultured cells are transfected withexpression plasmids of interest along with equal amounts of plasmidcontaining the promoter of interest linked to a reporter gene (e.g.GFP). As in Example 5 there are two methods by which the analyte ofinterest can be detected (directly and indirectly).

-   -   a) Direct immunoassay, in which an antibody specific to the        reporter is required. This antibody is labeled with an element        suitable for analysis by ICP-MS.    -   b) Indirect immunoassay, in which an antibody (primary antibody)        specific to the reporter is required. In addition a secondary        antibody specific to the primary antibody is labeled with an        element suitable for analysis by ICP-MS.        Cultured cells are grown and transfected with conditions of        interest. Upon analysis, the growth media is removed and the        cells are washed with 1× PBS three times. PBS is replaced with        ice-cold methanol and the culture dishes are incubated at        −20° C. for 5 minutes. The methanol is removed and the cells are        allowed to dry out completely. An assay buffer (e.g. 10% horse        serum, 1% BSA, 0.05% Tween-20, 1× PBS) is added to the culture        dishes and the dishes are incubated for 1-2 hours at room        temperature. In method a) an antibody specific to the reporter        is labeled with an element, diluted in dilution buffer and added        to the culture dishes. The cells are exposed to the antibody mix        for 2 hours at room temperature (or 37° C.). During this time,        the element-tagged antibody will bind the reporter. In method b)        the antibody specific to the reporter is not labeled. In both        cases, the un-reacted antibody is then washed away with wash        buffer. In method b) the element-labeled secondary antibody is        diluted in dilution buffer and applied to the cells. The dishes        are incubated for 1-2 hours at room temperature. The un-reacted        secondary antibody is then washed away with wash buffer.        Finally, in both methods, an acid solution (e.g. concentrated        HCl) is added, to release and dissolve the tagging element. The        dissolved element in acid is diluted with 10% HCl/1 ppb Ir to        provide an internal standard. The acid solution containing the        tagging element is analyzed by ICP-MS to quantify the protein of        interest.

Example 8 Detection of Proteins After Electrophoresis Using TaggedAntibodies

A sample of proteins is diluted in 2× SDS sample buffer (1% SDS, 2%glycerol, 1000 mM Tris, pH6.8, 5% β-mercaptoethanol, 1% DTT, 1% PMSF,0.2% leupeptin, 0.2% pepstatin) and exposed to electrophoresis on a 2-Dor polyacrylamide gel (SDS-PAGE or N-PAGE) to separate the proteins. Theproteins from the gel are transferred to nitrocellulose using a semi-dryelectrophoretic transfer apparatus (or equivalent). The nitrocelluloseis blocked for 1 hour at room temperature using a assay buffer (e.g. 5%milk in 1× PBS). An element-tagged antibody that recognizes the targetprotein is added to assay buffer and the nitrocellulose blot is exposedto the antibody-containing buffer for 2 hours at room temperature.Alternatively an un-labeled primary antibody that recognizes the targetprotein is used to bind the target protein, followed by washes with washbuffer, and then probing with a secondary anti-primary antibody that islabeled with an element. The nitrocellulose blot is washed three timeswith wash buffer (0.2% NP40 in 1× PBS). The protein in question isanalyzed and quantified by laser ablation.

Example 9 Detections of Proteins After Modification with 6× HIS-tag™(Invitrogen) and Separation by Electrophoresis

This Example is similar to Example 8; however, the proteins in thesample are modified prior to electrophoresis so that they have anaffinity for an element (e.g. the 6× HIS modification yields affinity toNickel). The gel or blotting paper containing the separated proteins iswashed with a solution containing an element (e.g. Ni) that is bound bythe protein modification. The gel or blotting paper is analyzed by laserablation (or direct excisions and elutions) and ICP-MS.

Example 10 Size Exclusion Gel Filtration Immunoassay

In this example, ICP-MS is used to detect the presence of a specificanalyte. Accordingly, an antibody is tagged with an element (eg. Au, Eu,Ru, etc.) and is introduced into a sample containing the analyte ofinterest. The elemental-tagged antibody reacts specifically to thetarget analyte. The resulting tagged analyte complex is separated fromun-reacted antibody using gel filtration (e.g. HiPrep Sephacryl™;Pharmacia) in a running buffer containing 1 ppbIr. The eluate iscollected in 0.5 ml increments into a 96 well plate, diluted in acid,and analyzed by ICP-MS.

Experimental results obtained according to this method for IgG analyteusing Fab′-Au antibody are shown in FIG. 5. In this experiment an IgGanalyte is incubated with an excess of Fab′-Au. The sample is runthrough a sephacryl S-200 column at a flow rate of 0.5 ml/min, using arunning buffer of 0.15M NaCl, 0.02M phosphate, pH 7.4, 1 ppb Ir. Thefigure provides the detector response as a function of elution time(eluate number). The first peak observed (the heavier molecular weight)corresponds to the reacted complex, having an expected molecular weightof about 235 kDa. The second peak corresponds to the unreacted taggedantibody having an expected molecular weight of about 85 kDa.

Example 11 Detection and Quantitation of Elemental Species

In this example, ICP mass spectrometry is used to measure a quantity ofmetal identified by an antibody which is specific for a given molecularform or species of a given metal. A solution containing the analyte isthen incubated with an antibody, which is specific for the molecularform of the given metal. This solution is treated to separateantibody-metal species complexes from un-reacted antibody and theremainder of components in the sample, although it is important onlythat species of the given metal other than the species of interest beremoved from the sample.

Preferably, the antibody exhibits little or no ability to bind tospecies other than the species of interest and exhibits a tight andspecific binding of the metal species which is to be measured.Preferably, this binding affinity shows an equilibrium dissociationconstant (K_(D)) on the order of 10⁻⁹ to 10⁻⁸M. The antibody used insuch assays also is able to resist interference from other componentscontained in the sample, which is being assayed. The solution containingthe antibody-metalspecies complexes is subject to standard ICP-MS/OESanalysis. This approach removes the necessity for a chromatographicpre-separation and consequently improves the sample integrity. It alsoallows for simultaneous measurement of several elemental species, themethod being limited only by the number of antibodies introduced to thesample.

Example 12 Detection and Quantitation of Elemental Species Using TaggedAntibodies

According to this Example, as in Example 11, antibodies specific formetal-species are raised according to methods well known to thoseskilled in the art. The difference in this Example is the antibody istagged with multiple atoms of a given tagging isotope, or astoichiometric mixture of isotopic tags. This has two potentialadvantages. First, in the event that the target metal element is subjectto interference in analysis through typical ICP-based interferences (forexample argide ion isobaric interferences) tagging the antibody with anormally non-interfered tag allows for interference-free determination,resulting in improved detectablity. Secondly, specific tags for variousspecies of the same target element allows simultaneous measurement ofvarious species (which would not be provided if the elemental tag werethe innate target element itself, since the presence of that element inthe spectrum would indicate only that one or more of the target speciesis present). A further advantage according to this approach is thattagging with multiple atoms of the same isotope allows for signalamplification proportional to the number of atoms of the same taggingisotope.

Example 13 Simultaneous Detection of Numerous Elemental Species in aSample Using Tagged or Untagged Antibodies

According to this example, as in Example 11 and 12, antibodies specificfor metalspecies are raised according to methods well known to thoseskilled in the art. The difference in this Example is that two or moreantibodies specific to different elemental species are incorporated, toallow for the simultaneous determination of different speciations of thesame or different elements (where each element is differentiallytagged).

Example 14 Immunoassay to Detect Bovine Spongiform Encephalopathy (BSE)in Animal Products

The methods of Examples 1, 2, 3, 4, 5, 8, and/or 10 are employed todetect BSE in animal products. There are several monoclonal antibodies(15B3, Korth et al., 1997; KG9, Laffling et al., 2001; Bio-RadLaboratories) that have been produced that target the prion protein PrPthought to be the infectious component responsible for the illness.Monoclonal antibodies specific to PrP are labeled with an element (eg.Au, Eu, Ru, etc.) and used in immunoassays described in either Example1, 2, 3, 4, 5, 8, and/or 10. Similar products known to be free of BSEwould be used as a negative control. In a similar manner other diseasesdetected for by antibody can be screened for (e.g. HIV, HTLV, Rabies,etc.).

Example 15 Immunoassay to Detect Ischemic Markers in Patients Believedto have Suffered a Heart Attack

The methods of Examples 1, 2, 3, 4, 5, 8, and/or 10 are employed tosimultaneously detect multiple ischemic markers in human samples.Candidate markers include: CK-MB, myoglobin, Troponin I, hsp70, BCL2,Bax, IGF, TNFα, angiostatin II.

Example 16 Method for Drug Discovery

In order to aid in drug discovery, animal cells or animal receptors areplaced in multi-well plates. The molecule of interest is added (i.e.potential drug), as well as element-tagged antibody (or element taggedligand) that recognizes the receptor. The potential drug is incompetition with the antibody for adhesion to the receptor. Unboundantibody is washed away, and the amount of bound antibody is determinedby ICP-MS. This is inversely proportional to the effectiveness of thepotential drug to recognize the receptor. If each well is provided withdifferently labeled antibodies, then by combining the contents of thewells, one can simultaneously assess the effectiveness of various drugs,or drug compositions by deconvoluting the resultant data. Likewise,differently labeled antibodies for the same analyte can be produced andplaced in corresponding wells of different plate (i.e. 10 differentlylabeled version of the antibody, each one placed in well 1, 1 in 10plates). The plate contents are combined, the reacted antibodies areseparated and analyzed simultaneously, with de-convolution to determinethe analyte concentration in the corresponding well of each plate.

Example 17 Detection of Tagged Proteins Using 2D Gel and MassSpectrometry

In this example, the ICP-DRC-MS technique is used in conjunction withthe laser ablation of polyacrylamide gels containing proteins tagged byiron. It is well known that ArN⁺ and ArO⁺ interfere with ⁵⁴Fe⁺ and⁵⁶Fe⁺, respectively. To facilitate the method described in Example 9, itis essential to remove isobaric poly-atomic interferences from the ironisotopes. For example, the ratio of the mass spectrometric signals atm/z=54:m/z=56 (where m/z indicates the mass-to-charge ratio of the ion)measured directly by ablation of the polyacrylamide gel containing aprotein band tagged by iron was found to be 1.14 (whereas the expectedvalue, based on the natural abundance of the iron isotopes, is 0.063).Utilizing ammonia as a reaction gas in the DRC environment, it ispossible to eliminate ArN⁺ and ArO⁺ interferences by charge transferreaction. This approach yielded the m/z=54:m/z=56 ratio thatapproximated the expected ⁵⁴Fe⁺/⁵⁶Fe⁺ isotope ratio, by which agreementthe determination of the tag iron can be confirmed. In addition, theprecision of this measurement is significantly improved due to partialtemporal equilibration of ions in the gaseous media of the reaction cell(see Bandura, D. R., et al. 2000).

Example 18 Detection of a Protein Using an Element-Tagged Aptamer

In this example, an element-tagged aptamer is used to detect and measurea protein in a sample.

Aptamers that bind a protein can be prepared using techniques known inthe art such as those described in Ellington A D and Szostak J W.(1990); Turek C and Gold L (1990); Robertson D L and Joyce G F (1990);Gold, L, Polisky, B, Uhlenbeck, O, and Yarus, M (1995); Szostak, J W(1995).

Aptamers may be labeled as is known to those skilled in the art.

A solution of the protein in the concentration range of 1000 to 0.5pg/μl in PBS buffer A is prepared. All dilutions are stored on ice.Subsequently, 100 μl of either protein dilution or PBS Buffer A (forcontrols) is pipetted into individual wells of a micro-titre plate (oreppendorf tubes). The element-tagged aptamer is pre-filtered through 300kDa centrifugal filter devices, such as MICRON™ or Centricon™). Serialdilutions of the tagged aptamer are prepared as is known to thoseskilled in the art. Depending on the concentration range of the protein,100 to 500 μl of the tagged aptamer is added to the wells or tubes andincubated for 1-2 hours at room temperature. Following incubation, thecombined sample is filtered in a centrifugal filter device as describedabove, for 15 minutes at 14,000 g at room temperature. The samplereservoir is inverted in a new vial and spun for 3 minutes at 1000 g totransfer the concentrate to a new vial. A fixed volume of the collectedprotein-aptamer mixture is diluted to 1 ml with 10% HCl/1 ppbIr forstabilization and analyzed in a mass spectrometer.

Example 19 Preparation of Kits Comprising Reagents for Cytokine Analysis

In this example, assay kits are prepared for the purpose of detectingand measuring either a single cytokine or multiple cytokinessimultaneously using an atomic mass or optical spectrometer. The kitsmay comprise reagents for (a) direct affinity assays, (2) sandwichaffinity assays, (3) competitive affinity assays, or any other assaysthat utilize element-tagged biologically active materials orelement-tagged analytes that are detected by an atomic mass or opticalspectrometer. The element-tagged affinity assays involve three basicsteps. First the target cytokine(s) from a sample (for example,EDTA-plasma) is bound to supports such as microwell plates, beads ormicrospheres. A solid support is not required if separation of reactionproducts (ie. bound tagged biologically active material from unboundtagged biologically active material) is achieved by chromatography (eg.size exclusion gel filtration or centrifugal filtration) instead of bywashing. This may be done using affinity, ionic or covalent bonding.Second, the target cytokine(s) is either complexed with anelement-tagged biologically active material (for example, anelement-tagged aptamer or antibody) or in the case of competitive assay,element-tagged cytokines are added to bind the remaining surfaceattachment sites. Third, after washing, the amount of element tag thathas been complexed is measured using an atomic mass or opticalspectrometer.

-   -   1) Preparation of the element-tagged biologically active        materials (for example, antibodies, aptamers) and element-tagged        competition analytes (for example, element-tagged cytokines) for        the kit. For direct or sandwich affinity assays, detection of        biologically active materials) (for example, antibodies or        aptamers) are raised against the cytokines of interest to be        measured. The cytokines of interest may include, Human IFNγ,        Human TNFα, Human IL-1β, Human IL-4, Human IL-5, Human IL-6,        Human IL-8, and Human IL-13. These detection antibodies or        aptamers are tagged with element tags. Preferably, they are        tagged in a covalent manner. For competitive assays, competition        analytes (for example, cytokines) are tagged with elemental        tags. Preferably, they are tagged in a covalent manner. It is        desirable to tag each pool of antibody, aptamer, or cytokine        with different elemental or isotopically enriched tags. For        example, anti-Human IFNγ-Sm¹⁵², anti-Human TNFα-Eu¹⁵¹,        anti-Human IL-1β-Dy¹⁶⁴, anti-Human IL-4-Eu¹⁵³, anti-Human        IL-5-Tb¹⁵⁹, anti-Human IL-6-Pr¹⁴¹, anti-Human IL-8-Sm¹⁵⁴, and        anti-Human IL-13-Gd¹⁵⁸ are prepared. To avoid tagging the active        sites of aptamers due to their small size, it may be necessary        to construct pre-labelled aptamer libraries, so that        element-tagged aptamers are selected and produced based on their        affinities to the respective cytokines. Optionally, if        element-tags are not found to interfere with affinities, it may        be easier to tag after the selection process. Both options are        available. Alternatively, the kits may simply include a tag        comprising a transition element with instructions for tagging        the biologically active material or competition analyte.        Preparation of tags comprising transition elements are known to        those skilled in the art.    -   2) Purification of element-tagged biologically active materials        (for example, antibody, aptamer) or element tagged competition        analytes (for example, cytokines). The element-tagged antibody,        aptamer or cytokine is purified preferably using size exclusion        chromatography, affinity chromatography, filtration, or        dialysis.    -   3) Several supports (for example, columns, gels, plates,        microwells and beads) have been designed for anchoring molecules        (for example, analytes, aptamers or antibodies) through for        example, the following mechanisms which include but are not        limited to: (a) hydrophobic interactions, (b) hydrophilic        interactions, (c) covalent reactions with amine and sulfhydryl        groups, (d) avidin interactions with biotynlated antibodies, (e)        Fc (crystallizable fragment of an antibody) affinities for        Protein A or Protein G, or (f) through affinities for nickel or        glutathione. Some examples of plates used for antibody        attachment are Nunc MaxiSorp™, Pierce Maleic Anhydride™, Pall™        nitrocellulose and PVDF™ filter plates. Aptamer attachment may        be achieved through several different methods, which includes        but is not limited to: UV irradiation, covalent chemical bonds,        or baking to glass, plastic, or membrane surface. Plates        available include: Nunc CovaLink™ which use secondary amino        groups to covalently bind 5′ carboxyl group of DNA or RNA; and        Nunc NucleoLink™ which covalently bind 5′ phosphorylated DNA.        Various filter-plates (Nunc™, Pall™) are available which are        convenient for preparing 96 well bead immunoassays        simultaneously. Such filter-plates can be used in conjunction        with vacuum or centrifuge and preferably have a pore size        1/10^(th) of the size of the beads being used. Other options of        bead containment are available and obvious to those skilled in        the art and can be used to substitute filter-plates in all        instances.    -   4) Coating of the microwell plates or beads with capture        anti-cytokine antibodies or aptamers. This can be done for the        sandwich affinity assays and the competitive affinity assays. In        the sandwich assay, preferably a matched pair of antibodies or a        matched pair of aptamers for each of the cytokines that are to        be quantitated is designed. A matched pair of antibodies is two        antibodies that recognize distinct epitopes on the cytokine so        that a cytokine may be bound with high affinity to both        antibodies simultaneously. The antibodies may be monoclonal        antibodies or polyclonal antibodies. A matched pair of aptamers        is similar, in that two aptamers are developed towards distinct        epitopes on the cytokine of interest so that the cytokine is        bound with high affinity to both aptamers simultaneously. In a        sandwich assay, one of the matched pair of antibodies or        aptamers is coated on the microwell plates or beads and serves        to capture the target cytokine in the sample anchoring it to the        surface. The second antibody or aptamer functions as a detection        agent and is labelled with an elemental tag. The coating of        microwell plates for multiple cytokine detection may be done        by a) pipetting a mixture of the capture antibodies or aptamers        into each well or b) by spotting different antibodies or        aptamers separately using different tips (e.g. robotic). The        advantage of a spotting format is that the different capture        antibodies or aptamers are kept separate and many different        custom plates can be produced for analyzing different        combinations of cytokines such that production is easily        designed by robotic application.    -   5) In sandwich assays and competition assays, after coating the        solid supports with capture molecules, for example antibodies or        aptamers to prevent non-specific binding, it may be necessary to        passivate or treat the microwell plates or beads and        filter-plate using a assay buffer prior to being used in the kit        protocol. The assay buffer may contain sterile proteinaceous        buffer (e.g. 1% BSA in 1× PBS with preservatives).    -   6) Components of Element-tagged Direct Affinity Assay kit for        the simultaneous detection and quantitation of multiple        cytokines. The kits may contain all or some of the following        components: 1) tags comprising transition elements, 2)        element-tagged biologically active materials (for example,        antibodies or aptamers), 3) cytokine binding solid supports, for        example microwell plate(s) or beads and filter-plate(s), 4)        standard cytokines (which will be comparable to the National        Institute for Biological Standards and Control (NIBSC)        standards), 5) diluent buffers for re-suspending standard        cytokines and element-tagged reagents, 6) wash buffers, 7)        elution buffers and 8) assay buffers (optional for pre-treating        plates), 9) instructions and protocols for direct affinity assay        for the simultaneous detection and quantitation multiple        analytes.    -   7) Components of Element-tagged Sandwich Affinity Assay kit for        the simultaneous detection and quantitation of multiple        cytokines. The kits may contain all or some of the following        components: 1)tags comprising transition elements, 2)        element-tagged biologically active materials (for example,        antibodies or aptamers), 3) pre-coated (with capture antibodies        or aptamers) and pre-treated (with assay buffer) solid supports,        for example microwell plate(s) or beads and filter-plate(s), 4)        standard cytokines (which will be comparable to NIBSC        standards), 5) diluent buffers for re-suspending standard        cytokines and element-tagged reagents, 6) wash buffers, 7)        elution buffers, and 8) assay buffers (optional for pre-treating        plates), 9) instructions and protocols for sandwich affinity        assay for the simultaneous detection and quantitation multiple        analytes.    -   8) Components of Element-tagged Competitive Affinity Assay kit        for the simultaneous detection and quantitation of multiple        cytokines. The kits may contain all or some of the following        components: 1) tags comprising transition elements; 2)        element-tagged competition analytes (for example, cytokines), 3)        pre-coated (with capture molecules, for example antibodies,        aptamers or other analyte binding material eg. maleic anhydride        groups or cytokine receptors) and pre-treated (with assay        buffer) solid supports, for example microwell plate(s) or beads        and filter-plate(s), 4) standard cytokines (which will be        comparable to NIBSC standards), 5) diluent buffers for        re-suspending standard cytokines and element-tagged reagents, 6)        wash buffers, 7) elution buffers and 8) assay buffers (optional        for pre-treating plates). 9) Protocols and instructions for the        competitive affinity assay. Optionally, the solid supports can        bind the analyte and competition analyte directly.

The kits may also include instructions or protocols for carrying out theassays.

The following are some sample protocols:

-   -   9) Protocol for Element tagged Direct Affinity Assay kit for the        simultaneous detection and quantitation of multiple cytokines.    -   a.) Prepare purified standard cytokines (which will be        comparable to NIBSC standards) by adding a prescribed volume of        standard cytokine diluent buffer (e.g. 1% BSA in 1×PBS) into        each vial, allowing to dissolve for 5 minutes at room        temperature, and inverting several times. Prepare serial        dilutions (e.g. 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml,        62.5 pg/ml, 31.2 pg/ml, 15.6 pg/ml, 7.8 pg/ml, 3.9 pg/ml, and 0        pg/ml).    -   b.) Prepare the sample containing cytokines of interest (eg.        patient EDTA-plasma samples) by thawing, mixing and filtering if        necessary.    -   c.) If beads are being used, beads must be suspended and diluted        accordingly. Aliquote 100 μl of beads into each microwell of        filter plate.    -   d.) Pipette an aliquote (eg. 100 μl) of either sample or        serially diluted standard into each well (in duplicate or        triplicate as desired). Shake on orbital shaker at room        temperature for 1-2 hrs.    -   e.) Aspirate (or with beads and filter-plate use centrifuge or        vacuum) and wash 3 times with 400 μl of wash buffer (e.g. 1% BSA        in 1× PBS).    -   f.) Add an aliquote (eg. 100 μl) of element-tagged biologically        active materials (for example, antibodies or aptamers) at        appropriate dilution (in detection diluent buffer; e.g. 1% BSA        in 1× PBS). Shake on orbital shaker at room temperature for 1-2        hrs.    -   g.) Aspirate (or with beads and filter-plate use centrifuge or        vacuum) and wash 3 times with 400 μl of wash buffer (e.g. 1% BSA        in 1× PBS).    -   h.) Aspirate (or with beads and filter-plate use centrifuge or        vacuum) plate.    -   i.) Add 50-100 μl elution buffer containing an acid solution        with elemental spike, preferably an element that is soluble in        the acid and close to the same atomic mass as the elements to be        measured (e.g. 3% HCl, 1 ppb Pr). Shake on orbital shaker at        room temperature for 5-10 minutes.    -   j.) Measure and quantitate elements of interest using atomic        mass or optical spectrometer, preferably ICP-MS.    -   10) Protocol for Element tagged Sandwich Affinity Assay kit for        the simultaneous detection and quantitation of multiple        cytokines.    -   a.) Prepare purified standard cytokines (which will be        comparable to NIBSC standards) by adding a prescribed volume of        standard cytokine diluent buffer (e.g. 1% BSA in 1× PBS) into        each vial, allowing to dissolve for 5 minutes at room        temperature, and inverting several times. Prepare serial        dilutions (e.g. 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml,        62.5 pg/ml, 31.2 pg/ml, 15.6 pg/ml, 7.8 pg/ml, 3.9 pg/ml, and 0        pg/ml).    -   b.) Prepare the sample containing cytokines of interest (eg.        patient EDTA-plasma samples) by thawing, mixing and filtering if        necessary.    -   c.) If beads are being used, beads must be suspended and diluted        accordingly. Aliquote 100 μl of beads into each microwell of        filter plate.    -   d.) Pre-treat microwell plate or beads and filter-plate with 400        μl of assay buffer (e.g. 1% BSA in 1× PBS) per well. Shake on        orbital shaker at room temperature for 1-2 hrs. (optional, may        not be necessary)

e.) Pipette an aliquote (eg. 100 μl) of either sample or seriallydiluted standard into each well (in duplicate or triplicate as desired).Shake on orbital shaker at room temperature for 1-2 hrs.

-   -   f.) Aspirate (or with beads and filter-plate use centrifuge or        vacuum) and wash 3 times with 400 μl of wash buffer (e.g. 1% BSA        in 1× PBS).    -   g.) Add an aliquote (eg. 100 μl ) of element-tagged biologically        active materials (antibodies or aptamers) at appropriate        dilution (in diluent buffer; e.g. 1% BSA in 1× PBS). Shake on        orbital shaker at room temperature for 1-2 hrs.    -   h.) Aspirate (or with beads and filter-plate use centrifuge or        vacuum) and wash 3 times with 400 μl of wash buffer (e.g. 1% BSA        in 1× PBS).    -   i.) Aspirate (or with beads and filter-plate use centrifuge or        vacuum) plate.    -   j.) Add 50-100 μl elution buffer containing an acid solution        with elemental spike, preferably an element that is soluble in        the acid and close to the same atomic mass as the elements to be        measured (e.g. 3% HCl, 1 ppb Pr). Shake on orbital shaker at        room temperature for 5-10 minutes.    -   k.) Measure and quantitate elements of interest using atomic        mass or optical spectrometer.    -   11) Protocol for Element tagged Competitive Affinity Assay kit        for the simultaneous detection and quantitation of multiple        cytokines.    -   a.) Prepare purified standard cytokines (which will be        comparable to NIBSC standards) by adding a prescribed volume of        standard cytokine diluent buffer (e.g. 1% BSA in 1× PBS) into        each vial, allowing to dissolve for 5 minutes at room        temperature, and inverting several times. Prepare serial        dilutions (e.g. 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml,        62.5 pg/ml, 31.2 pg/ml, 15.6 pg/ml, 7.8 pg/ml, 3.9 pg/ml, and 0        pg/ml).    -   b.) Prepare the sample containing cytokines of interest (eg.        patient EDTA-plasma samples) by thawing, mixing and filtering if        necessary.    -   c.) If beads are being used, beads must be suspended and diluted        accordingly. Aliquote 100 μl of beads into each microwell of        filter plate.    -   d.) Pre-treat microwell plate or beads and filter-plate with 400        μl of assay buffer (e.g. 1% BSA in 1× PBS) per well. Optionally,        shake on orbital shaker at room temperature for 1-2 hrs.    -   e.) Pipette an aliquote (eg. 100 μl) of either sample or        serially diluted standard into each well (in duplicate or        triplicate as desired). Shake on orbital shaker at room        temperature for 1-2 hrs.    -   f.) Aspirate (or with beads and filter-plate use centrifuge or        vacuum) and wash 3 times with 400 μl of wash buffer (e.g. 1% BSA        in 1× PBS).    -   g.) Add an aliquote (eg. 100 μl) of element-tagged competition        analyte (for example, cytokines) at appropriate dilution (in        diluent buffer; e.g. 1% BSA in 1× PBS). Shake on orbital shaker        at room temperature for 1-2 hrs.    -   h.) Aspirate (or with beads and filter-plate use centrifuge or        vacuum) and wash 3 times with 400 μl of wash buffer (e.g. 1% BSA        in 1× PBS).    -   i.) Aspirate (or with beads and filter-plate use centrifuge or        vacuum) plate.    -   j.) Add 50-100 μl elution buffer containing an acid solution        with elemental spike, preferably an element that is soluble in        the acid and close to the same atomic mass as the elements to be        measured (e.g. 3% HCl, 1 ppb Pr). Shake on orbital shaker at        room temperature for 5-10 minutes.    -   k.) Measure and quantitate elements of interest using atomic        mass or optical spectrometer.

The kits may include two or more biologically active materials havingdistinguishable elemental tags for simultaneous determination of two ormore analytes. In the case of kits for competition assays, the kits mayinclude two or more competition analytes having distinguishableelemental tags and corresponding two or more capture molecules for thesimultaneous determination of two or more analytes. Finally, the kitsmay also include a combination of components for the direct affinityassay, the sandwich affinity assay and the competitive affinity assay.For example, a tagged competition analyte is used to measure analyte Aby a competition affinity assay and two differently tagged biologicallyactive materials are used to measure analytes B and C by a directaffinity assay.

Example 20 Cytokine Immunoassay

The following provides an example of the methods of the invention usingelement tags to determine the concentration of an analyte (eg. cytokine)of interest in a complex sample (FIGS. 6-11). Either Nanogold-Fab′™ oranother element-labeled Fab′ (or other biologically active material)that will bind specifically to the target analyte (or primary,secondary, or tertiary antibody) may be used. There are three types ofimmunoassays that may be used:

-   -   a) Direct immunoassay, which would involve trapping the        protein(s) (Human IgG, FLAG-BAP, FIG. 6) or cytokine(s) of        interest (Human Interferon gamma, IFN-g; Human Interleukin 5,        IL-5, Human Interleukin 6, IL-6, and Human Interleukin 8, IL-8,        FIG. 7) using a specified solid support such as microwell plates        or microspheres (Maleic Anhydride microwell plates were used in        experiment shown in FIG. 6 and Pall NT Acrowell Filter microwell        plates were used in the experiment shown in FIG. 7), incubating        with an excess of element-tagged (anti-Human IgG Fab′-nanoAu,        FIG. 6; anti-IFN-.gamma.-Sm, anti-IL-5-Eu, anti-IL-6-Tb, and        anti-IL-8-Dy, FIG. 7) or untagged primary antibody or other        biologically active material (anti-BAP, FIG. 6) specific to the        target analyte, washing off the un-reacted antibody, adding        element-tagged secondary antibody (anti-mouse-Eu, FIG. 6),        washing off un-reacted antibody and then subjecting the        antibody-analyte complexes to atomic mass or optical        spectrometry (eg. ICP-MS).    -   b) Sandwich immunoassay which would involve trapping the        cytokine(s) of interest (IL-6 in FIG. 8; IL-6 and IL-8 in FIG.        9; IL-6, IL-8, IFN-.gamma. and TNF-.alpha. in FIG. 10; and IL-6        and TNF-.alpha. in FIG. 11) by incubating the sample containing        the cytokine(s) of interest with a specified solid support such        as the microwell plates (FIGS. 8-10) or microspheres (FIG. 11)        that have been pretreated (bound) with an excess of primary        antibody or biologically active material specific to the target        analyte. The microwell plates or microspheres are washed to        remove the un-reacted portion of the sample, and the cytokine is        then exposed to element-tagged or untagged biologically active        material (eg. primary antibody), washed to remove unreacted        biologically active material, and if necessary incubated with        element-tagged secondary antibody. The washed cytokine complexes        are then subjected to atomic mass or optical spectrometry (eg.        ICP-MS).    -   c) Competition immunoassay, which would involve trapping the        target cytokine of interest (protein X) by incubating a        specified solid support (eg. microwell plates or microspheres        which may or may not be pretreated with an excess of primary        antibody or biologically active material specific to the target        analyte), adding the analyte-containing sample, washing off the        unbound components, and then exposing the specified support with        antibody-protein X complexes to a known amount of purified        element-labeled protein X. The washed cytokine complexes are        then subjected to atomic mass or optical spectrometry (eg.        ICP-MS).        PBS buffer A is prepared as follows: 150 mM NaCl; 20 mM        phosphate, pH 7.4; 1% BSA (bovine serum albumin). All eppendorf        tubes, micro-titer plates, and filters, and slurry of        microspheres to be used subsequently are treated with PBS buffer        A for 1 hour at room temperature to block non-specific binding.        This treatment will reduce the non-specific interactions that        occur between the plastic used and the analyte and        Nanogold-Fab′™. Alternatively low retention plastic products can        be used (e.g. Axygen tubes). Following this a solution of the        analyte (cytokine, peptide, protein, etc.) in a specified        concentration range (eg. 1-100 ng/ml in FIG. 6; 0.1-10 ng/ml in        FIG. 7; 3.12-300 pg/ml IL-6 in FIGS. 8, 9 and 10; 31.2-2000        pg/ml IL-8 in FIG. 9 and 10; 15.6-1000 pg/ml of IFN.gamma. and        TNF-.alpha. in FIG. 10), 16-10,000 pg/ml of TNF-.alpha. and IL-6        in FIG. 11) in PBS buffer A or biological matrix (eg. EDTA        plasma) is prepared. All dilutions are stored on ice.        Subsequently, 100 ul of analyte or either PBS buffer A or other        biological matrix such as EDTA-plasma (for negative controls) is        pipetted into the individual wells of the micro-titer plate (or        set of eppendorf tubes). If using Nanogold-Fab′™ to remove large        unbound gold particles, the Nanogold-Fab′™ is pre-filtered        through 300 KDa MICRCON™ (or Centricon™) centifugal filter        devices. Filtered Nanogold-Fab′™ (and other element-tagged and        untagged biologically active materials, eg. in FIG. 6,        anti-Human IgG-Fab′-nanoAu, anti-BAP, anti-mouse-Eu; in FIG. 7,        anti-IFN-.gamma.-Sm, anti-IL-5-Eu, anti-IL-6-Tb, and        anti-IL-8-Dy; in FIG. 8, anti-IL-6-Tb; in FIG. 9, anti-IL-6-Tb,        anti-IL-8-Dy; in FIG. 10, anti-IFN-.gamma.-Sm,        anti-TNF-.alpha.-Eu, anti-IL-6-Tb, and anti-IL-8-Dy; and in FIG.        11, anti-TNF-.alpha.-Eu and anti-IL-6-Tb) required are diluted        in PBS buffer A or other biological matrix such as EDTA-plasma        are prepared as follows: a 1:50 dilution is produced by adding        60 μl of Nanogold-Fab′™ in 2940 μl PBS buffer A. A 1:500        dilution is then produced by adding 100 μ of 1:50 Nanogold-Fab′™        to 900 μl of PBS buffer A. In methods a) and b) depending on        concentration range of analyte, 100 to 500 μl of 1:500 (or other        suitable dilution) of Nanogold-Fab′™ (or other required        element-tagged or untagged biologically active materials) is        then added to the wells of the plate and then incubated for 1-2        hours at room temperature. In method c) an element-tagged        analyte (cytokine) is added to each sample. In methods a-c)        microwell plates are washed by pipetting fresh wash buffer in        and out of wells 1-3 times. Microspheres in filter plates are        washed through centrifugation of wash buffer (1-3 times).        Microspheres in tubes are washed through centrifugation of wash        buffer (1-3 times). Preferably all procedures are at 4 C. If an        untagged biologically active material has been used in the        proceeding step, the sample may be incubated with an        element-tagged biologically active material (eg. secondary        antibody) to bind the cytokine complex. The wash step will then        need to be repeated. If an untagged biologically active material        has been used in the proceeding step, the sample may be        incubated with an element-tagged biologically active material        (eg. tertiary antibody) to bind the cytokine complex. The wash        step will then need to be repeated. Finally, a fixed volume of        10%HCl/1 ppbIr is added to each well. Ir and/or Ho provides an        internal standard for ICP-MS quantitation and the acid solution        is suitable for the elemental analysis.

Experimental results obtained according to method a) are given in FIGS.6 and 7, using Human IgG and FLAG-BAP (FIG. 6) and IFN-.gamma., IL-5,Il-6, and IL-8 (FIG. 7) as the analytes of interest with anti-Human IgGF′ab-Au, anti-BAP and anti-mouse-Eu (FIG. 6), and anti-IFN-.gamma.-Sm,anti-IL-5-Eu, anti-IL-6-Tb, and anti-Il-8-Dy as the (un)tagged antibody(FIG. 7). Experimental results obtained according to method b) are givenin FIGS. 8-11.

In FIG. 6, Maleic Anhydride microwell plates (Pierce) were used toanchor the proteins to the bottom of each of the 96 wells. Wells intriplicate contained concentrations of both Human IgG and FLAG-BAP ineither 0, 1, 10 or 100 ng/ml in PBS buffer A. According to method a)Human IgG was bound with anti-Human IgG Fab′-nanoAu and FLAG-BAP wasbound with anti-BAP and anti-mouse-Eu antibodies. Detection andquantitation was obtained using an ICP-MS.

In FIG. 7, Pall Acrowell filter plates were used to anchor the proteinsto the bottom of each of the 96 wells. Wells in triplicate containedconcentrations of both IFN-.gamma,. IL-5, IL-6, and IL-8 in either 0,0.1, 1, or 10 ng/ml in PBS buffer A and according to method a) thesehuman cytokines were bound with anti-IFN-.gamma.-Sm, anti-IL-5-Eu,anti-IL-6-Tb, and anti-IL-8-Dy antibodies respecitively. Detection andquantitation was obtained using an ICP-MS.

In FIG. 8-10, Quantikine microwell plates (R&D systems) were used toanchor the proteins (cytokines) to the bottom of each of these 96 wells.Wells in triplicate contained concentrations of (as shown in graphs aswell as 0 pg/ml for negative control) in PBS buffer A (or EDTA-plasma)and according to method b) these human cytokines were bound withspecific element tagged antibodies (see FIGS. 8-10). Detection andquantitation was obtained using an ICP-MS.

In FIG. 11, Fluorokine microspheres (R&D systems) were used to anchorthe proteins (cytokines) and supplied filter plates were used to containand perform each assay. Wells in triplicate contained concentrations of(as shown in graphs, as well as 0 pg/ml for negative control) in PBSbuffer A and according to method b) these human cytokines were boundwith specific element tagged antibodies (see FIG. 11). Detection andquantitation was obtained using an ICP-MS.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents, and parent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or parent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

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We claim:
 1. A method for the detection and measurement of a transition element in a sample, where the measured transition element is a tag on an aptamer that binds with an analyte, comprising: a) combining a tagged aptamer with the analyte, where the tagged aptamer binds with the analyte; b) separating bound tagged aptamer from unbound tagged aptamer; and c) detecting and measuring the transition element by an atomic mass or optical spectrometer having a source of ions or atomic ions.
 2. A method for the detection and measurement of an element in a sample, where the measured element is a tag on an aptamer that binds with an analyte, comprising: a) combining the aptamer with the analyte; b) introducing a transition element to the combined aptamer and analyte, wherein the transition element binds with the aptamer; and c) detecting and measuring the transition element by an atomic or optical spectrometer having a source of ions or atomic ions.
 3. A method for the detection and measurement of an element in a sample, where the measured element is a tag on a competition analyte, comprising: a) combining a tagged competition analyte with at least one of an analyte and analyte complex, where the tagged competition analyte and at least one of the analyte and anlayte complex are in competition for a binding site; b) separating bound tagged competition analyte from the unbound tagged competition analyte; and c) detecting and measuring the transition element on the bound competition analyte by an atomic mass or optical spectrometer having a source of atoms or atomic ions, wherein the detection and measurement of the transition element is related to the detection and measurement of at least one of the analyte and analyte complex.
 4. The method of claim 3 wherein the binding site is located on a capture molecule.
 5. A method for the detection and measurement of a positively charged transition element in a sample, where the measured transition element is a specific tag which is unnaturally bound on a biologically active affinity material, wherein the biologically active affinity material binds with an analyte or analyte complex, comprising: i) combining the tagged biologically active affinity material with the analyte or analyte complex, where the tagged biologically active affinity material binds with the analyte or analyte complex; ii) separating the analyte or analyte complex which is bound to the tagged biologically active affinity material from unbound tagged material; and iii) detecting and measuring the positively charged transition element by one of an inductively coupled plasma mass spectrometer and an inductively coupled plasma optical emission spectrometer wherein said transition element is any element having an atomic number of 21-29, 39-47, 57-79 or
 89. 6. The method of claim 5 wherein the step ii) includes a step of electrophoresis of the analyte or analyte complex to separate the analyte or analyte complex which is bound to the tagged biologically active affinity material from the unbound tagged material.
 7. The method of claim 5 wherein the analyte complex comprises a primary biologically active affinity material and an analyte, and wherein the tagged biologically active affinity material is a secondary biologically active affinity material tagged with the transition element.
 8. The method of claim 5 wherein the tagged biologically active affinity material is an biologically active affinity material tagged with a nanoparticle having a metal core of 50-70 gold atoms.
 9. The method of claim 5 wherein the step of detecting and measuring the positively charged transition element includes an inductively coupled plasma mass spectrometer.
 10. The method of claim 5 wherein the transition element is an isotope.
 11. The method of claim 5 wherein the transition element is selected from a group consisting of the noble metals, lanthanides, rare earth elements, gold, silver, platinum, rhodium, iridium and palladium.
 12. The method of claim 5 wherein the transition element is gold, and wherein the gold has a diameter of 3 nm or less.
 13. The method of claim 5 wherein the tagged biologically active affinity material comprises a covalent coupling of the transition element to the biologically active affinity material.
 14. The method according to claim 5 wherein the biologically active affinity material is selected from a group consisting of an aptamer, antigen, hormone, growth factor, receptor, protein and nucleic acid.
 15. The method of claim 5 wherein the tag is selected from the group consisting of a plurality of elements, a plurality of isotopes, a plurality of atoms of an isotope, a different number of atoms of each isotope, and combinations thereof, and wherein the step of detecting and measuring comprises detecting and measuring the transition element free of the analyte or analyte complex.
 16. The method of claim 15 comprising an additional step of introducing two or more biologically active affinity materials or analytes having distinguishable elemental tags into a sample of interest for simultaneous determination, and wherein the step of detecting and measuring comprises detecting and measuring the transition element free of the analyte or analyte complex.
 17. The method of claim 5 comprising an additional step of sample introduction to one of the atomic mass and optical spectrometer, wherein the sample introduction includes laser ablation.
 18. The method of claim 5 which further comprises a step of running the analyte or analyte complex on an electrophoresis gel prior to the step of combining the tagged biologically active affinity material with the analyte or analyte complex, to isolate the analyte or analyte complex from the sample.
 19. A method for the multiplexed detection and measurement of positively charged transition elements in a sample, wherein the measured transition elements are specific tags, each of which is unnaturally bound on a biologically active affinity material, and wherein the biologically active affinity material binds with an analyte or analyte complex, comprising: i) providing a plurality of biologically active affinity materials, wherein each biologically active affinity material of said plurality is tagged with at least one positively charged transition element in a manner that permits a given biologically active affinity material to be distinguished from other biologically active affinity materials of said plurality by an inductively coupled plasma spectrometer; ii) combining said materials with a plurality of analytes and/or analyte complexes, wherein said materials bind with the analyte or analyte complexes in a manner that permits a given analyte or analyte complex to be distinguished from other analytes or analyte complexes by an inductively coupled plasma spectrometer; iii) separating the analytes and/or analyte complexes that are bound to the tagged biologically active affinity materials from unbound tagged materials, and then iv) detecting and measuring, either simultaneously or sequentially, the positively charged transition elements with an inductively coupled plasma mass spectrometer or an inductively coupled plasma optical emission spectrometer, wherein said transition element is any element having an atomic number of 21-29, 39-47, 57-79 or
 89. 20. The method according to claim 19, comprising detecting and measuring with an inductively coupled plasma mass spectrometer.
 21. A method according to claim 19, wherein the positively charged transition elements comprise one or more isotopes of positively charged transition elements.
 22. A method according to claim 19, wherein the step of detecting and measuring further comprises distinguishing one isotope of a transition element from one or more different isotopes of the same element or from one or more different elements.
 23. The method of claim 19 comprising an additional step of introducing two or more biologically active affinity materials or analytes having distinguishable elemental tags into a sample of interest for simultaneous determination, and wherein the step of detecting and measuring comprises detecting and measuring the transition element free of the analyte or analyte complex.
 24. The method of claim 19 wherein the biologically active affinity material is selected from the group consisting of an aptamer, antigen, hormone, growth factor, receptor, protein and nucleic acid.
 25. The method of claim 5 wherein the analyte is a cytokine. 